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E-Gel® Technical Guide - Thermo Fisher Scientific

1. esee tnenens 30 Results with E Gel Single Comp Gels 24 eer a pun md nein tte e Met 33 Results with E Gel Double Comb Gels ccsssssssecssssseseeesesessesecescscscecsceacseseeesceacacseecceacseececceacseeseesecaceeeeeeseeaeess 35 Troubles NOO juko ica pr 36 Electrophoresis of E Gel NGS and E Gel with SYBR Safe Gels eeeeeeeeeeeeeeeeee nennen 37 Sample RES AU A Ch ON ons ese cca snc A Bes denoted tates equos O T o alas ocv emet buie 97 Molecular Weight Markers sse petet edite edes pa ie te ecentesdiee toil d eme bati tesi O fevte tod edunt eud 38 Using E Gel NGS and E Gel with SYBR Safe Gels with the iBase Power System see 39 Visualizing E Gel NGS and E Gel with SYBR Safe Agarose Gel3S ccsssssssssessteteteseseseneneneseaeseseaeaeeeseaeerees 43 Results using E Gel with SYBR Safe Agarose Gels sess nene tenetntntnnnnns 44 POMP VSS INO Ot eet aii cu ed us E as Scant e anuncia S octaua tte cet bte bte ur ae 46 iii Electrophoresis Using E Gel EX Agarose Gels s ecccsssseeeeeeeeeneeeeeeseeneeeeeseneeeseeseeneeeeenseeneeeeeseeeeeenss 48 Sample l reparaloliscossdes ud ud obersten a rReE Te CRC berto rere er eer meter ae ere ern cere Cae Orr tactu een ere 48 Molecular Were hb Markers ce eco E ode devi paie saa aeisen teeta dete Qual eet ater delete leta uta ro dettes eu s 49 Using E Gel EX Agarose Gels with the iBase Power System 50 Ru
2. Melted gel Increased current due to Do not run the gel longer than 15 minutes longer run times Sample leaking Sample is overloaded Load the recommended sample volume per well from the wells Use the Two Step Loading method page 117 Wells damaged during comb Remove the comb gently without damaging the wells removal 57 Troubleshooting Continued Observation RNA sample cannot be seen Failure Mode indicated by continuous rapid beeping and Cassette Missing Hold Go to Reset or a steady red light Speckles visible High background suboptimal or no image Stripes visible on image Low cloning efficiency 58 Cause Inhibition of visualization by heat and denaturing agent Defective cassette Cold cassette or improper operating conditions Dust fluorescing in same wavelength as SYBR Safe No filters or wrong filter set Photographic settings not optimal No IR coating on camera when using an UV system Used a UV light source to visualize DNA Solution Wait 10 15 minutes for gel to cool before visualization Disconnect the base and replace gel cassette with a fresh gel cassette Press and release the power button or Go button for 2 seconds to return to Ready Mode Use a cassette stored at room temperature Avoid storing gel cassettes at 4 C Use E Gel iBase at room temperature 20 25 C Make sure gel is clean before imaging Refer to page 1
3. Continue holding the Go button and insert the power plug into the electrical outlet Then connect the cable to the iBase device Release the Go button and connect the iBase device to the computer with a USB A to B cable A into the computer B end into the iBase The computer should now begin to search for the iBase This step may take several minutes The program indicates that it is searching for the iBase The program indicates that the iBase device has been found Press Next to begin the iBase Firmware Update Do not disconnect or use the device until the update is complete Once the update is complete the program will indicate that the update was successful 10 Disconnect the USB cable from the iBase device 11 The iBase device is now updated In case a message The Update Failed Retry the program and if the problem persists contact Technical Support see page 110 for further assistance Parameters for E Gel iBase Programs Introduction The E Gel iBase Power System contains a number of different programs to run different types of E Gel agarose gels Refer to the table below for the run parameters default time and maximum allowable time for each program Program Default Run E Gel 0 8 1 2 2 4 0 E Gel double comb 0 8 2 E Gel CloneWell 0 8 1 E Gel 0 8 1 2 2 4 2 E Gel 496 3 E Gel double comb 0 896 296 4 E Gel CloneWell 0 8 5 E Gel Clo
4. on an E Gel Base 132 Instructions are provided below to perform electrophoresis of E Gel with SYBR Safe E Gel single comb gels and double comb gels with the E Gel Base previously available from Invitrogen Note You will need a power supply for electrophoresis with an E Gel Base You must first pre run the E Gel agarose gel for 2 minutes with the comb in place before loading your samples to ensure proper resolution of your DNA fragments Each E Gel cassette is supplied individually wrapped and ready for use To set up and use an E Gel gel follow the instructions below 1 Open the package containing the gel and insert the gel with the comb in place into the apparatus right edge first Press firmly at the top and bottom to seat the gel in the base You should hear a snap when it is in place The Invitrogen logo should be located at the bottom of the base close to the positive pole See the diagram below Power Supply comb wells underneath Red Bottom Top Connect electrical leads from the base unit to the power supply Pre run the gel with the comb in place for 1 2 minutes at 60 70 V or 40 50 mA Do not exceed 2 minutes Turn off the power supply Gently remove the comb from the gel Load the samples in the wells of the E Gel as described on page 27 Proceed to running the gel below Run the gel at 60 70 volts constant voltage or 40 50 mA constant current for
5. v 4 is controlled by two buttons on top of the base The left button is for a double comb run and right button is for a single comb run see the label on the unit To select different electrophoresis runs for the PowerBase do one of the following page 25 for details e Press and release the button run OR e Press and hold the button pre run electrical outlet je d light adaptor RC buttons NC dl Top Bottom E Gel Base The E Gel Base see figure below previously available from Invitrogen connects to a power supply and is used for electrophoresis of E Gel single comb and double comb agarose gels page 130 for details Power Supply l comb wells Red eee underneath Top Bottom E Gel Accessories E Gel Opener Cleaning and storage Gel Knife Cleaning and storage 10 The E Gel Opener is an easy to use device specifically designed to open any E Gel single comb double comb or E Gel with SYBR Safe cassette for staining excision of DNA fragments or for blotting The E Gel Opener consists of an anodized aluminum platform housing two recessed steel blades one which is stationary and one which is movable The blades are brought into contact with the E Gel cassette by turning the large knob clockwise Blade Table edge After use clean the E Gel Opener with mild detergent and water to remove any excess agarose ethidium bromide and plastic from the platfo
6. Enter the appropriate time listed under Run Time from Reference Line to Collection Well from the Run Time Estimation Table see page 76 for your band Press Go to run the gel Monitor the run carefully As the run ends the band of interest may be seen migrating into the collection well p UR LB a Bands that have entered the Collection Well Collect DNA from the wells using a pipette without piercing the bottom of the well Proceed to your application using the collected DNA If the band of interest has overshot the collection well use the Reverse E Gel program to run the band back into the collection well Additional DNA bands can be collected from the same well s Be sure to refill the collection wells with more water as water is lost during the run Collecting DNA Using E Gel SizeSelect Agarose Gels Continued Speed run using the iBase Power System Interrupt a run on the iBase Power System Run in reverse direction using the iBase Power System The iBase device is pre programmed with a SPEED E Gel program for performing runs using high power to generate rapid yes no results The program is suitable for 0 8 1 2 and 2 E Gels only This program is limited to 7 minutes where the bands migrate less than half the length of the gel A run exceeding 7 minutes under these conditions results in a defective run This mode is not compatible with E Gel 476 gels Electrophoresis can be interrupted at any time
7. Imaging gels containing SYBR Safe gel stain Important Note Exposure time and gain setting Disposal of gels containing SYBR Safe DNA gel stain E Gel NGS and E Gel with SYBR Safe gels contain the SYBR Safe DNA gel stain and do not have to be stained after electrophoresis The SYBR Safe DNA gel stain has a fluorescence excitation maxima at 280 and 502 nm and an emission maximum at 530 nm when bound to nucleic acids Use a blue light or UV light transilluminator to view the gel a filter is required to photograph the gel your standard ethidium bromide filter may not be appropriate View E Gel NGS and E Gel with SYBR Safe gels using these instruments e Blue light transilluminator The E Gel Safe Imager Real time Transilluminator and Safe Imager 2 0 Blue Light Transilluminator Cat nos G6500 and G6600 are designed specifically for use with SYBR Safe stained DNA gels Refer to page 17 for instructions on using the E Gel Safe Imager Real time Transilluminator or Safe Imager 2 0 Blue Light Transilluminator Blue light transilluminators available from other manufacturers are also compatible for use with E Gel NGS and E Gel with SYBR Safe gels e Standard 300 nm UV transilluminator e Imaging systems such as laser based scanners equipped with an excitation source in the UV range or between 470 530 nm Note If you plan to excise bands for cloning use a blue light transilluminator to visualize
8. Safe DNA gel stain the proprietary stain in E Gel EX and E Gel SizeSelect gels and many of ony AND a AD SO DD our other nucleic acid and Wavelength nm protein stains such as SYBR Gold SYBR Green I and II SYPRO Ruby SYPRO Orange and Coomassie Fluor Orange S un un Tul Em Unlike UV transilluminators the E Gel Safe Imager Real time Transilluminator does not produce UV light and does not require UV protective equipment during use Blue light transillumination also results in dramatically increased cloning efficiencies compared to UV transillumination The E Gel Safe Imager Real time Transilluminator cannot be used for viewing ethidium bromide stained gels 18 Safe Imager Blue Light Transilluminators Continued Safety information The E Gel Safe Imager Real time Transilluminator is an electrical device for the E Gel Safe e Never touch the power cord or outlet with wet hands Imager Real time e Do not use this device in damp areas or while standing on damp floors Transilluminator e Do not attempt to open the E Gel Safe Imager Real time Transilluminator e Use the E Gel Safe Imager Real time Transilluminator with the power cord supplied your starter kit or with the E Gel iBase Power System This power cord has a universal transformer compatible with 90 V to 220 V Only these power cords should be used to power the device Attach the power cord to the E Gel
9. Safe Imager Real time Transilluminator at the back of the device Plug the other end of the power cord into a properly grounded electrical outlet ensuring the correct plug adaptor is attached e Always disconnect the E Gel Safe Imager Real time Transilluminator from electrical outlet before cleaning device e Donotleave the E Gel Safe Imager Real time Transilluminator switched on for extended periods of time Always switch the unit off after use Always use the E Gel Safe Imager amber filter unit or E Gel Safe Imager viewing glasses to protect your eyes while viewing gels The E Gel Safe Imager Real time Transilluminator does not produce UV light however it does emit an intense blue light Published literature has identified blue light as a possible risk factor for macular degeneration although no clinical studies have been published Note The amber filter unit will NOT protect your eyes when viewing gels on UV transilluminators and although the viewing glasses do block UV light they are not designed for use as UV safety glasses Viewing gels with 1 Place the Amber filter unit on top of the sample as shown below or use the viewing the E Gel Safe glasses when excising bands from DNA gels 2 Switch the E Gel Safe Imager Real time Transilluminator on using the ON OFF button in one of these ways e Toturnon the light for 30 seconds press and release the ON OFF button The LED indicator light will be a flashing gre
10. increments Press and hold the time increases button continuously automatically stops at 00 109 Accessory Products E Gel Agarose Gels Product E Gel CloneWell Gels E Gel CloneWell 0 8 SYBR Safe Gels 18 Pak E Gel CloneWell 0 8 SYBR Safe Gel and iBase Starter Kit E Gel Single Comb Gels E Gel 1 2 with SYBR Safe Starter Kit E Gel 1 2 with SYBR Safe E Gel 2 with SYBR Safe Starter Kit E Gel 2 with SYBR Safe E Gel EX 1 Starter Kit E Gel EX 1 10 Pak E Gel EX 1 20 Pak E Gel EX 2 Starter Kit E Gel EX 2 10 Pak E Gel EX 2 20 Pak E Gel EX 4 10 Pak E Gel NGS 0 8 10 Pak E Gel NGS 0 8 Starter Kit E Gel SizeSelect 2 10 Pak E Gel SizeSelect 2 Starter Kit E Gel 0 8 with Ethidium Bromide Starter Pak E Gel 0 8 with Ethidium Bromide 18 Pak E Gel 1 2 with Ethidium Bromide Starter Pak E Gel 1 2 with Ethidium Bromide 18 Pak E Gel 2 with Ethidium Bromide Starter Pak E Gel 2 with Ethidium Bromide 18 Pak E Gel 4 with Ethidium Bromide 18 Pak E Gel Double Comb Gels E Gel 0 8 double comb with Ethidium Bromide 18 Pak E Gel 2 double comb with Ethidium Bromide 18 Pak 110 Appendix A Quantity 18 gels 18 gels E Gel iBase Power System Safe Imager Blue Light Transilluminator and E Gel High Range DNA Marker 6 gels and E Gel PowerBase 18 gels 6 gels and E Gel PowerBase 18 gels 10 gels E Gel iBase P
11. mee Daughter E Base The E Holder Platform is designed to hold E Gel 96 gels during robotic loading Use the E Holder when you need to load multiple gels on a robotic platform while the other gels are running on the E Base Note The E Holder is not a power supply unit cannot be connected to an electrical outlet and cannot be used to run gels The E Editor 2 02 software allows you to quickly reconfigure digital images of E Gel 48 or 96 gel results for analysis and documentation Capture an image of the gel and then use the E Editor 2 02 software to e Align and arrange the lanes in the image e Save the reconfigured image for further analysis e Copy and paste selected lanes or the entire image into other applications for printing saving e mailing and or publishing on the Web The E Editor 2 02 software can be downloaded for free at www lifetechnologies com egels and following the instructions to download the software and user manual Safe Imager Blue Light Transilluminators Advantages of blue light transillumination Instrument specifications A Caution Introduction Unlike UV transilluminators Safe Imager transilluminators do not produce UV light which result in the following advantages The Safe Imager does not require UV protective equipment during use Blue light transillumination results in dramatically increased cloning efficiencies compared to UV transillumination Safe
12. 1 ng band of DNA Fast and easy purification of DNA fragments 50 bp 2 000 bp in size Compatibility with blue light transillumination to dramatically reduce DNA damage and maximize cloning efficiency Low Throughput E Gel Electrophoresis System Low throughput E Gel electrophoresis system System components Applications The following components are available for low throughput electrophoresis E Gel CloneWell E Gel with SYBR Safe E Gel EX E Gel NGS E Gel SizeSelect E Gel single comb and E Gel double comb pre cast agarose gels Gels are available in a variety of percentages Choose an appropriate gel based on your application see table on page 2 E Gel iBase Power System and E Gel PowerBase v 4 The E Gel iBase Power System and PowerBase v 4 are a base and a power supply in one device These power systems connect directly to an electrical outlet using the adaptor supplied with the base page 7 E Gel Safe Imager Real time Transilluminator This transilluminator emits blue light and is specifically designed for use with SYBR Safe stained DNA gels run on the E Gel iBase Power System page 17 E Gel Opener The E Gel Opener is an implement specifically designed to open any E Gel single comb double comb or E Gel with SYBR Safe gel cassette page 10 Gel Knife The Gel Knife is used to open E Gel EX and E Gel NGS cassettes page 10 The Low Throughput E Gel Electrophores
13. Can be used for separation of RNA Cassette easily opened with gel knife E Gel NGS pre cast agarose gels contain SYBR Safe DNA gel stain and have the following features Low agarose concentration for resolution of large fragments for NGS and cloning applications Contain SYBR Safe DNA gel stain instead of ethidium bromide Compatibility with blue light transillumination to dramatically reduce DNA damage and maximize cloning efficiency E Gel with SYBR Safe pre cast agarose gels have the following features Contain SYBR Safe DNA gel stain instead of ethidium bromide to o Minimize generation of hazardous waste SYBR Safe DNA gel stain is not classified as hazardous under US Federal regulations o Reduce exposure to the strong mutagen ethidium bromide and UV exposure Compatibility with blue light transillumination to dramatically reduce DNA damage and maximize cloning efficiency E Gel CloneWell pre cast agarose gels have the following features Fast and easy purification of DNA fragments 100 bp 6 000 bp in size DNA fragments easily recovered from the gel using a pipette Contain SYBR Safe DNA gel stain instead of ethidium bromide Compatibility with blue light transillumination to dramatically reduce DNA damage and maximize cloning efficiency E Gel SizeSelect pre cast agarose gels have the following features Contain a proprietary fluorescent nucleic acid stain High sensitivity with detection down to
14. During electrophoresis samples migrate between the wells of the row below For example the bands of the lane B11 migrate between well C11 and C12 see figure on the next page This configuration provides a 1 6 cm run length allowing resolution between 100 bp and 10 kb The staggered well format of the gel cassette is compatible with automated liquid handling devices that use 8 12 or 96 tip loaders During sample loading the samples will fall onto the slopes of the wells and be drawn into the wells by capillary force A diagram of the E Gel 96 cassette is shown below For details on the gel see previous page J Copper electrode i ZLI Sample migration E op OEC CB C P IP BIA between wells j E Gel 96 Invitrogen me hehe Copper electrode barcode E Base Power Supply E Base Mother E Base Two types of bases are available from Life Technologies The Mother E Base Cat no EB MO3 has an electrical plug that can be connected directly to an electrical outlet and is used for electrophoresis of one E Gel 48 E Gel 96 E PAGE 48 or E PAGE 96 gels The Daughter E Base Cat no EB D03 connects to the Mother E Base and together they can be used for the electrophoresis of two or more E Gel 48 E Gel 96 E PAGE 48 or E PAGE 96 gels Note The Daughter E Base does not have an electrical plug and cannot be used without a Mother E Base See next page
15. E a roe Terre Te re ee errr vi DH EEO CEE ON baee 1 EA hal exer 0 0 Ore SiE O S 2 0 ee ey rr et en eT Rr ECT en E ee ee Qe 1 Cel SEIS CHO nesia E E 2 Low Ihroughput E Gel Electrophoresis Dy Sese dero edu oie aA 5 Low Throughput E Ge Well Formats iren usted bue rel e dais aqu uda te ai beste tu a iiieehs 6 Eel ibase DONE SUED oos tte i uet aeons ee 7 Ie gU ip e 9 E d vut c oio Met 10 Medium Throughput E Gel Electrophoresis System essent ene nenennnetntnenns 11 Ier m V M IC 12 High Throughput E Gel Electrophoresis System asusta beer dabat sett tired Go e netted 13 PCer CI e M eae panera 14 Es Base Powet ou DDD orateio xoti poplite itae doboteat uid catuli ani oet aped ult are cauli reer 15 Sate Imager Blue Light Transilluminators 595i eti qoe inet sitet bua e tlie ui cu 17 Product SDSciHieatloBs so sev a E A T E T O E N 22 Method T 25 General Gudenes sisi a a E a R ticus Oba iE DbedetUE 25 Electrophoresis of E Gel Agarose Gels ccsssseccsssseeeeeesseeeeesenseceesenseneeesensneneeesenseneeesesseneeesensenenenenss 27 Sample FE Pata Ol liceasusnsnsccssnnessusugeqcainsaen tusneasecasetenequstya eastanasa an aeatqareanaeemeganaesaataia te muatarsateraea yao aan 27 Moleculat Weig NEMT Ke Sosi E aedes entis edic aceasta daneben dude uei undis 28 Using E Gel Agarose Gels with the iBase Power System
16. Gel Sample Loading Buffer page 112 Run gels stored at room temperature Keep samples uniform and load deionized water into empty wells Load gel within 15 minutes of opening the pouch Run gel within 1 minute of loading samples E Gel agarose gels can only be used once Do not re run E Gel agarose gels that have already been used DNA samples are loaded in E Gel agarose gels using a One Step Loading or Two Step Loading method The One Step Loading method is the standard method for loading E Gel agarose gels The Two Step Loading method is an optional method that is only necessary if the One Step Loading method produces fuzzy or indistinct bands or the gel has been removed from its plastic pouch for an extended period of time see Appendix page 117 for details Loading buffer is optional Samples can be loaded directly into the wells if no loading buffer is used If you are using loading buffer mix the required amount of DNA with the loading buffer We recommend using a loading buffer with the following formulation in its final concentration E Gel agarose gels E Gel CloneWell and SizeSelect gels e 10mM Tris HCl pH 7 5 e 10mM Tris HCl pH 7 5 e 1mM EDTA e 1mM EDTA e 0 005 bromophenol blue e 0 005 xylene cyanol FF If using 10X BlueJuice Gel Loading Buffer or TrackIt Loading Buffer see page 110 dilute this buffer 50 to 200 fold to obtain optimal results with E Gel agarose gels 25 General Guidelin
17. Gel Opener are extremely sharp Do not insert your fingers into the area housing the blades Pick up the E Gel Opener by holding the large knob only see Figure 1 above Exercise caution when handling and cleaning the E Gel Opener Dispose of blades in a needle disposal container or a Sharps disposal box 119 E Gel Opener Continued Opening an E Gel The following section provides instructions to open an E Gel cassette Before beginning single comb or you should wear safety goggles and gloves double comb i cassette Place the E Gel Opener on a flat surface with the knob extending off the edge of the laboratory bench and facing the user Set the E Gel Opener to its widest open position by turning the knob counterclockwise Insert the E Gel into the E Gel Opener so that two opposing sides of the gel cassette are aligned with the blades see Figure 1 Position the E Gel such that the two sides fit into the grooves housing the blades Turn the knob steadily clockwise to bring the blades in contact with the E Gel cassette As the knob is tightened you will hear a series of pops Continue to turn the knob until the resistance increases Stop turning the knob as soon as the E Gel cassette begins to lift off the surface of the platform Two sides of the E Gel will now be unsealed Note Once you observe the E Gel cassette begins to lift off the surface of the platform do not continue to tighten the knob as you will
18. Note Supercoiled DNA molecular weight markers may produce a slightly fuzzy pattern when run on E Gel NGS and E Gel with SYBR Safe agarose gels E Gel 1 296 with SYBR Safe E Gel 296 with SYBR Safe E Gel NGS 38 E Gel 1 Kb Plus DNA Ladder E Gel High Range DNA Marker 100 bp DNA Ladder Kb Plus DNA Ladder High DNA Mass Ladder E Gel 1 Kb Plus DNA Ladder E Gel Low Range Quantitative DNA Marker 25 bp DNA Ladder 50 bp DNA Ladder 100 bp DNA Ladder Low DNA Mass Ladder E Gel 1 Kb Plus DNA Ladder E Gel High Range DNA Marker 1 Kb Plus DNA Ladder 500 bp DNA Ladder High DNA Mass Ladder TrackIt 1 Kb Plus DNA Ladder 10488 090 12352 019 15628 019 10787 018 10496 016 10488 090 12373 031 10597 011 10416 014 15628 019 10068 013 10488 090 12352 019 10787 018 10594 018 10496 016 10488 085 Load 500 750 ng markers ina volume of 20 uL Load 100 250 ng markers in a volume of 20 uL Using E Gel NGS and E Gel with SYBR Safe Gels with the iBase Power System Introduction After preparing your samples proceed with electrophoresis Instructions are provided below to load and run E Gel NGS and E Gel with SYBR Safe gels using the E Gel iBase Power System E Gel with SYBR Safe gels but not E Gel NGS gels are compatible with the E Gel PowerBase v 4 and E Gel Base For details on using E Gel agarose gels with the E Gel PowerBase v 4 see page 130 For details on using E Ge
19. Refer to page 127 to determine the optimal filter sets to use or contact the instrument manufacturer for advice Optimize settings of your system for E Gel SizeSelect agarose gels empirically You may need to increase the exposure time or gain setting Use IR blocking filter or emission filter with IR coating Use Reverse program as described in the E Gel Technical Guide to run band back into collection well Fill the second row of wells with sterile water prior to running your band of interest into the wells Collect DNA from the well in two or more fractions refill with water after each collection Load the recommended amount of DNA Load the recommended amount of DNA View gel in darkened room or use 50 bp ladder as reference marker Refer to Run Time Table to determine when to collect the sample 83 Electrophoresis of E Gel 48 96 Gels General Guidelines for E Gel 48 96 Gels Introduction Note Materials Needed DNA Sample Capacity E Gel 48 and E Gel 96 gels are designed for medium and high throughput electrophoresis of DNA fragments using the Mother E Base and Daughter E Base For optimal results follow the guidelines for preparing your DNA sample as described in this section e The E Gel 48 and 96 gels can only be used once Do not re use e The E Gel 48 and 96 gels are compatible with the E Gel 96 mother base and daughter base available previously from Invitrogen For instru
20. and loading method For proper band separation we recommend keeping sample volumes uniform Load deionized water or TE into any empty wells Gel was not electrophoresed For best results run the gel within 1 minute of sample immediately after sample loading loading Expired gel used Use properly stored gels before the expiration date Longer electrophoresis run Longer run times cause an increase in the current resulting time or high current during in poor band migration or a melted gel Do not run the gel the run longer than recommended time for each gel type Melted gel Increased current due to Do not run the gel longer than 25 minutes longer run times Sample leaking Sample is overloaded Load the recommended sample volume per well from the wells Use the Two Step Loading method page 117 Wells damaged during comb Remove the comb gently without damaging the wells removal 82 Troubleshooting Continued Observation High background suboptimal or no image Stripes visible on image Band of interest below collection well Low volume in collection well Low yield bands smeared Low yield bands not visible Cause No filters or wrong filter set Photographic settings not optimal No IR coating on camera when using an UV system Run time too long Well not refilled prior to collection Excess quantity of DNA Low quantity of DNA Solution
21. by pressing and releasing the Go button to stop the current A flashing red light indicates that the current is stopped and the digital display flashes the message Press GO to Run Hold Go to Reset to indicate that the run was interrupted You can remove the gel from the iBase device to check the progress of the run then e Continue the run from the point at which it was stopped Reinsert the gel and press and release the Go button The light changes to a steady green and the LCD display shows the count down time The run time but not the program can be adjusted before continuing the run e Cancel the interrupted run Press and hold the Go button for a few seconds The LCD display resets and returns to Ready Mode A new program and run time can be selected to rerun the gel The E Gel iBase Power System is pre programmed with a program to run E Gel agarose gels in a reverse direction 1 Toggle between program minutes and seconds by pressing the Mode button until the program blinks 2 Select the REVERSE E Gel Program using the Up Down A V buttons to change the program 3 Tochange the run time press the Mode button until the minutes or seconds blink and change the values using the Up Down buttons the maximal run time for reverse running is 3 minutes 4 Press the Go button yo start electrophoresis a green light indicates that the run is in progress The LCD displays the count down time while the run is in prog
22. cassette must be in contact with the two electrode connections on the iBase device The LED produces a steady red light to indicate that the cassette is correctly inserted Slide cassette into electrodes Press left side to secure 3 Remove the comb and load your samples Be sure to load molecular weight markers and add water to any empty wells Note It is not necessary to pre run E Gel NGS or E Gel with SYBR Safe agarose gels Load the E Gel agarose gel within 15 minutes of opening the pouch and run the gel within 1 minute of loading samples Avoid introducing bubbles while loading as bubbles will cause bands to distort 1 Remove the comb from the E Gel NGS or E Gel with SYBR Safe gel using both hands to lift the comb gently by rolling the comb slowly towards you Be careful to pull the comb straight up from both sides Do not bend the comb Remove any excess fluid using a pipette Load 20 uL of sample per sample well see page 37 for details Load 20 uL 500 700 ng of the appropriate molecular weight markers page 38 Load 20 uL of water into any remaining empty wells Using E Gel NGS and E Gel with SYBR Safe Gels with the iBase Power System Continued Electrophoresis using the iBase Power System Speed run using the iBase Power System Toggle between program minutes and seconds on the iBase device by pressing the Mode button until the program blinks Use the Up Down A V buttons
23. damage For optimal results follow the guidelines for preparing your DNA sample as described in this section Note For instructions to run E Gel 96 with SYBR Safe gels refer to the chapter Electrophoresis of E Gel 48 96 Gels page 84 DNA sample Molecular weight markers page 38 Optional E Gel Sample Loading Buffer page 112 Use 20 100 ng DNA per band for samples containing one unique band or up to 500 ng per lane E Gel NGS and E Gel 1 2 with SYBR Safe or 700 ng per lane E Gel 2 with SYBR Safe of samples containing multiple bands If you are unsure how much to use test a range of concentrations to determine the optimal concentration for your particular sample Excess DNA will cause poor resolution The recommended total sample volume for E Gel NGS and E Gel with SYBR Safe is 20 uL Note For best results keep all sample volumes uniform If you do not have enough samples to load all the wells of the gel load an identical volume of deionized water into any empty wells Prepare your samples by adding deionized water to the required amount of DNA to bring the total sample volume to 20 uL For samples that are in a high salt buffer refer to page 26 Loading buffer is optional See page 25 for more details 37 Molecular Weight Markers We recommend using the following DNA molecular weight markers for different types of E Gel agarose gels to obtain good resolution DNA molecular weight markers
24. damage the E Gel Unscrew the knob and remove the E Gel The E Gel cassette fits snugly in the recessed groove and you may have to carefully work the cassette from the housing Turn the E Gel 90 and re insert the gel cassette into the Opener so that the two remaining sealed sides can be opened Repeat Step 2 to open the remaining two sides of the E Gel Stop turning the knob when you see the top of the E Gel cassette begins to lift off the gel Unscrew the knob and carefully remove the E Gel cassette The 4 sides of the cassette should be unsealed If not repeat Steps 2 5 as necessary Remove the E Gel and set the opened cassette on your bench If you plan to blot the gel do not pick up the gel from the cassette Lift off the top of the gel cassette Place the blotting membrane on the gel and pick up the cassette with the gel and membrane Flip the gel and membrane out of the cassette onto your gloved hand and then flip the gel and the membrane directly onto your wet blotting paper If you plan to purify DNA from the gel lift off the top of the gel cassette and excise the gel fragment Transfer the gel slice to a microcentrifuge tube Discard E Gel agarose gels with ethidium bromide as hazardous waste SYBR Safe stain is not classified as hazardous waste under US Federal regulations but contact your safety office for appropriate disposal methods Cleaning and After use clean the E Gel Opener with mild detergent and wate
25. dedil kc scien deadewal reci ea UD caidubleulsdiauustsbnlnueiodeiauaeaoadeumteandonses 106 Usine B Gel 96 Mother Daughter Base sa ieu ir sedebit istaec E dixe edel 106 E Gel 96 Mother Daughter base Quick Reference Guilde torto o bte ets ert base e ae natu te usta ed 108 PED PON GDA EE 110 Accessory TD EOCBGIS coegit Sv da D NE NA IM MEME MM UI M S NES se du Se 110 Technical Sup POEE anisina nadteniel slept an Sita uel Eco ot tete te buenos a die eolit its relie testen Sosa ansedaats ds 113 Appendix B Sal Gly canis i D Ie dRI II AMI NEN NE MIU IN M NEM M I MI MINI E 114 Explanator of Symbols anid Warnunpses sisti decepit iota rad Cp t aptent esr presion toti a ipae amita n teta a postu 114 Appendix Two Step Loading Protocoles ien iuo ip Eaa a VE UEUNNEEEEE IAEEES A RS EDU E UE 117 Two Step Loading ot E Gel Ae Arse Gelaren DU IHR R iR iu RS Ru ae 117 Appendix D Openine E Gel Cassettes cene icut exce ce auod a RE Vus 119 ig er OP D 119 Opening E Gel EX and E Gel NGS Agarose Gel Cassettes sss entente 121 Appendix E Nucleic Acid Gel Stains for E Gel Agarose Gels eeeeeeeeeeeseeeeeeee nennen 123 SYDR Safe DNA CES iae ui netta utet ca etr eet M ei E 124 Proprietary Fluorescent Nucleic Acid Gel SUM ood esters sete NONI assem MI ON Na Ad es Do erae e ese RN 126 Fiter Selechon Guides tetti ione ditiis ote S afatetudicsse A inu Mdpsdubunpa t CRM UM Cade 127
26. down time e To cancel the rest of the interrupted run press and hold the pwr prg button for a few seconds The digital display will reset and the base will return to Ready Mode If desired you can then program a new run time as described on page 89 and rerun the gel In case of an external power failure loss of electricity or the electrical cord is accidentally removed from the outlet the run will continue when the power resumes The Mother E Base or Daughter E Base will signal the end of the run as described on the previous page except the light will be an alternating red green to indicate that an external power failure had occurred during the run The surfaces of the Mother E Base and Daughter E Base should be kept free of contaminants To clean disconnect bases from power source and wipe clean with a dry cloth Do not attempt to open the Mother E Base or Daughter E Base To honor the warranty bases should only be opened and serviced by Life Technologies E Base Quick Reference Guide A quick reference guide for operating the Mother E Base and Daughter E Base is provided below Operating modes and electrophoresis runs are described Mode Action Sound Light Digital Display Introduction Base plugged in Ready with no current flowing through gel End of run Run ends after an external power failure Pause manually end the run Mother E Base connected to an electrical outlet Gel casse
27. figure below c e Normalized fluorescence excitation and S Excitation amp emission spectra of SYBR Safe DNA gel MEL E stain determined in the presence of DNA Q Q d z E E 300 400 500 600 700 Wavelength nm Visualization of Detect DNA bands stained with SYBR Safe DNA gel stain using a blue light SYBR Safe transilluminator a standard UV transilluminator or a laser based scanner For photographing gels a special filter may be required refer to page 127 for more information Cloning benefits of By using the blue light transillumination for visualization DNA damage is dramatically SYBR Safe reduced thus improving cloning efficiency For more information go to www lifetechnologies com us en home life science dna rna purification analysis nucleic acid gel electrophoresis dna stains sybr safe html 125 Proprietary Fluorescent Nucleic Acid Gel Stain Introduction Disposal of E Gel EX and E Gel SizeSelect agarose gels Spectrum of proprietary fluorescent nucleic acid gel stain Visualization of proprietary fluorescent nucleic acid gel stain Cloning benefits of proprietary fluorescent nucleic acid gel stain 126 A proprietary nucleic acid stain has been specifically developed for E Gel EX and E Gel SizeSelect This gel stain has high sensitivity with detection down to 1 ng band of DNA In addition this proprietary fluorescent nucleic acid stain can be viewed by blue light trans
28. gel E Gel with SYBR Safe Dilute samples with loading E Gel EX gel buffer deionized water or TE E Gel NGS gel to a final volume of 20 uL E Gel SizeSelect gel E Gel 96 gel E Gel 48 gel Dilute samples with loading buffer deionized water or TE to a final volume of 15 uL Electrophoresis of E Gel Agarose Gels Sample Preparation Introduction Materials needed Amount of DNA Total sample volume Preparing samples Loading buffer For optimal results follow the guidelines for preparing your DNA sample as described in this section DNA sample Molecular weight markers page 28 Optional E Gel Sample Loading Buffer page 112 Use 20 100 ng DNA per band for samples containing one unique band or up to 500 ng per lane for samples containing multiple bands If you are unsure how much to use test a range of concentrations to determine the optimal concentration for your particular sample Excess DNA will cause poor resolution The recommended total sample volume for each gel type is listed in the table below Note For best results keep all sample volumes uniform If you do not have enough samples to load all the wells of the gel load an identical volume of deionized water into any empty wells Gel Type Total Sample Volume E Gel single comb gel 20 uL E Gel double comb gel 20 uL Prepare your samples by adding deionized water to the required amount of DNA to bring the total sample volume to 20 uL F
29. of SYBR Safe DNA gel stain produces no signs of mortality or toxicity at a limit dose of 5000 mg kg e Visualizing E Gel with SYBR Safe using blue light transilluminators dramatically reduces DNA damage that lowers cloning efficiency For details on SYBR Safe DNA gel stain see page 123 Features of The proprietary fluorescent nucleic acid stain in E Gel EX and E Gel SizeSelect pre proprietary cast agarose gels offer the following advantages fluorescent nucleic 9 Detection sensitivity to 1 ng band of DNA acid gel stain e Compatibility with blue light transillumination to reduce DNA damage that lowers cloning efficiency 123 SYBR Safe DNA Gel Stain Introduction Safety features of SYBR Safe Disposal of SYBR Safe 124 SYBR Safe DNA gel stain has been specifically developed for reduced mutagenicity making it safer than ethidium bromide for staining DNA in agarose gels The detection sensitivity of E Gel with SYBR Safe stain is similar to that of E Gel containing ethidium bromide DNA bands stained with SYBR Safe DNA gel stain can be detected using a standard UV transilluminator a visible light transilluminator or a laser based scanner e SYBR Safe DNA gel stain is not classified as hazardous waste under US Federal regulations e SYBR Safe stain meets the requirements of the Clean Water Act and the National Pollutant Discharge Elimination System regulations though E Gel with SYBR Safe generall
30. of sample into each well Load 20 uL of sample buffer containing the same salt concentration as your sample into any remaining empty wells Load the appropriate DNA markers page 85 in the marker M wells of an E Gel 48 and 96 gels 90 Running E Gel 48and 96 Gels Using E Base Instructions for running E Gel 48 and E Gel 96 gels in a Mother E Base or Daughter E Base are provided below Note It is not necessary to have a gel in the Mother E Base if you are using a Daughter E Base However the Mother E Base must be plugged into an electrical outlet 1 To begin electrophoresis press and release the pwr prg button located on the lower right corner of the Mother E Base The red light will change to a green light and the digital display will show the count down time while the run is in progress to electrical outlet electrode connection ERI E s barcode electrode EE wSx connection power button Mother E Base digital timer light LED display If you are using a Daughter E Base press and release the pwr prg button located on the lower right corner of the Daughter E Base Mother E Base 9 4 p E 3 To add to the run time while the run is in progress press the time button to select the desired time and then release the time button To interrupt or stop a run in progress see next page 2 The Mother E Base or Daughter E Base will signal the end of the run with a flashing
31. page 117 Expired gel used Use properly stored gels before the expiration date Longer electrophoresis run Longer run times cause an increase in the current resulting in time or high current during poor band migration or a melted gel Do not run the gel longer the run than recommended time for each gel type Melted gel Increased current due to Do not run the gel longer than 40 minutes longer run times Sample leaking Sample is overloaded Load the recommended sample volume per well from the wells Use Use the Two Step Loading method page117 si Two Step Use the Two Step Loading method page117 si method page 117 Wells damaged during comb Remove the comb gently without damaging the wells removal 46 Troubleshooting Continued Problem Failure Mode indicated by continuous rapid beeping and Cassette Missing Hold Go to Reset or a steady red light Speckles visible High background suboptimal or no image Stripes visible on image Low cloning efficiency Cause Defective cassette Cold cassette or improper operating conditions Dust fluorescing in same wavelength as SYBR Safe No filters or wrong filter set Photographic settings not optimal No IR coating on camera when using an UV system Used a UV light source to visualize DNA Solution Disconnect the base and replace gel cassette with a fresh gel cassette Press and release the power button or Go butt
32. page 28 Toggle between program minutes and seconds on the E Gel iBase by pressing the Mode button until the program blinks Use the Up Down A V buttons to select the appropriate program for your gel Gel Type Program Default Run Maximal Run Time Time E Gel 0 8 1 2 2 RUN E Gel 0 8 2 0 26 minutes 40 minutes E Gel 4 RUN E Gel 4 30 minutes 40 minutes E Gel double comb 0 8 2 RUN E Gel DC 13 minutes 20 minutes The SPEED E Gel program is available for 0 8 1 2 and 2 E Gels see page 32 To change the run time press the Mode button until the minutes or seconds blink Use the Up Down buttons to change the values up to the maximal run time Press the Go button to start electrophoresis a green light indicates that the run is in progress The LCD displays the count down time while the run is in progress The device stops automatically when the programmed time has elapsed A flashing red light and beeping rapid beeping for 30 seconds followed by a single beep every minute signals the end of the run The LCD displays Run Complete Press Go Press and release the Go button to stop the beeping The LED shows a steady red light and the LCD display shows the most recent program and settings Remove the E Gel cassette from the E Gel iBase and proceed to imaging or other application with the gel To open the E Gel cassette for staining excision of DNA fragments or for blotting see page 119 for
33. red light and rapid beeping for 2 minutes followed by a single beep every minute At the end of the run the digital display will show the original time setting not any time change that was made during the electrophoresis The digital display will also show the elapsed time up to 19 minutes with a negative sign since the end of the run 3 Press and release the pwr prg button to stop the beeping The light will turn to a steady red and the digital display will show the last time setting 4 Remove the gel cassette from the Mother E Base or Daughter E Base You are now ready to capture an image of the gel Note The bands in the gel will diffuse within 20 40 minutes 91 Running E Gel 48and 96 Gels Continued Note Interrupting an Electrophoresis Run Maintaining E Base M 92 We recommend that you disconnect the Mother E Base from the electrical outlet when not in use for a prolonged period of time You can interrupt an electrophoresis run at any time by pressing and releasing the pwr prg button to stop the current The stopped current is indicated by a steady red light and the digital display will flash to indicate that the run was interrupted You can remove the gel from the E Base to check the progress of the run Then e To continue the run from the point at which it was stopped reinsert the gel and press and release the pwr prg button The light changes to steady green and the digital display shows the count
34. sets to use suboptimal or no or contact the instrument manufacturer for advice a SYBR Sate Photographic settings not Optimize settings of your system for E Gel with SYBR gel optimal Safe empirically You may need to increase the exposure time or gain setting Stripes visible on No IR coating on camera Use IR blocking filter or emission filter with IR coating image SYBR Safe when using an UV system gel 105 Appendix Using E Gel 96 Mother Daughter Base Introduction Instructions are provided below to perform electrophoresis of E Gel 48 and 96 gels with E Gel 96 mother base and daughter base previously available from Invitrogen Note The E Gel 96 mother base and daughter base are designed differently than Mother and Daughter E Base For instructions on using the Mother E Base and Daughter E Base see page 91 Using E Gel 96 The recommended run time for E Gel 48 gels is 20 minutes and E Gel 96 gels is 12 Mother Base and minutes ia Base 1 Connect the electrical plug from the E Gel 96 mother base to an appropriate electrical outlet 110 V or 220 V If a gel cassette is not inserted the light on the mother base is not illuminated If you are using an E Gel 96 daughter base connect the daughter base to a mother base 2 A red light at the lower left corner of the mother base and daughter base will illuminate when the E Gel 48 or 96 cassette is correctly inserted and the digital d
35. the following components e E Gel 48 gels see below and next page e Mother E Base and Daughter E Base page 15 e E Editor 2 02 Software page 16 Applications E Gel 48 agarose gels are suitable for analyzing multiple samples e PCR products e Restriction digests e RI PCRreactions e Primer dimers 4 E Gel 48 gels e Library screenings e Diced dsRNA 4 E Gel 48 gels e SNPs analysis 11 E Gel 48 Agarose Gels E Gel 48 Gels Separation range for E Gel 48 Gels 12 E Gel 48 gels are self contained pre cast agarose gels that include agarose a proprietary buffer system ethidium bromide and electrodes packaged inside a dry disposable UV transparent cassette Each E Gel 48 gel contains 48 sample lanes and 4 marker lanes and is designed for medium throughput agarose electrophoresis of nucleic acids This configuration provides a 3 2 cm run length See page 22 for product specifications The 4 6 E Gel 48 gels are prepared with high resolution agarose to ensure quality resolution of DNA fragments below 400 bp see next page for separation range The wells of the E Gel 48 gel are compatible for loading with a multichannel pipettor The lane numbers are labeled with fluorescent dye that transfers to the image and allows tracking of your samples during photo documentation of the gel In addition each E Gel 48 cassette is labeled with an individual barcode to facilitate identification of the gel using commercial
36. upper and lower IEM see page 6 and well areas with the Gel Knife 6 If you plan to purify DNA from the gel excise the gel fragment Transfer the gel slice to a microcentrifuge tube 122 Appendix E Nucleic Acid Gel Stains for E Gel Agarose Gels Available nucleic E Gel agarose gels come in four different formats for staining your DNA acid gel stains e Regular E Gel agarose gels contain the standard DNA gel stain ethidium bromide e E Gel with SYBR Safe contains SYBR Safe DNA gel stain which is not classified as hazardous waste under US Federal regulations and improves cloning efficiency when using blue light for imaging e E Gel EX and E Gel SizeSelect agarose gels contain a proprietary fluorescent nucleic acid stain compatible with blue light visualization for increased nucleic acid detection sensitivity Advantages of SYBR Safe DNA gel stain is a safer more environmentally friendly alternative to SYBR Safe DNA ethidium bromide and offers the following advantages gel stain e SYBR Safe DNA gel stain is not classified as hazardous waste under US Federal regulations and meets the requirements of the Clean Water Act and the National Pollutant Discharge Elimination System regulations e SYBR Safe DNA gel stain does not cause mutations chromosomal aberrations or transformations in appropriate mammalian test systems in contrast to ethidium bromide which is a strong mutagen e Asingle oral administration
37. you to reconfigure digital images of E Gel 48 and E Gel 96 gels for analysis and documentation The staggered lanes in an E Gel 96 gels are difficult to compare and analyze by standard 1 D gel analysis programs such as Bio Rad s Quantity One Phoretix 1D or Kodak 1D software E Editor 2 02 software reconfigures the wells of an E Gel 48 and E Gel 96 gel into a side by side format for easy comparison and analysis You can reconfigure gels that were scanned in the original gel cassette or gels that were removed from the cassette You can also group the images of multiple gels loaded from a 384 well microtiter plate into a single image with a layout corresponding to that of the original plate Capture an image of the gel as described below and then use the E Editor 2 02 software to e Align and arrange the lanes in the image e Save the reconfigured image for further analysis e Copy and paste selected lanes or the entire reconfigured image into other applications for printing saving e mailing and or publishing on the Web Imaging the Gel Use an appropriate gel documentation system to capture a digital image of the gel When imaging the gel should be properly aligned i e not at an angle and gel features should be clear and distinct Proceed to Downloading Software Downloading E Editor 2 02 software can be downloaded for free from Software www lifetechnologies com egels and follow the instructions to download the software and us
38. 0 3kb PCR fragment 11 250 bp DNA Ladder 400 ng 12 High DNA Mass Ladder 200 ng 2 Agarose GP 2 3234 5 8 7T B8 9 10 11 12 O CON BD OTF W NY 33 Results with E Gel Single Comb Gels Continued 2 single comb gel Results obtained using a 2 agarose gel are shown below using 20 uL per lane Digestion of pUC18 and pcDNA 3 1 are as described on the previous page Lane Sample Low DNA Mass Ladder 250 ng 100 bp DNA Ladder 300 ng pcDNA 3 1 Nco I cut 150 ng pcDNA 3 1 uncut 120 ng pUC18 Pst I 60 ng 50 bp DNA Ladder 300 ng 50 bp DNA Ladder 300 ng 450 bp PCR fragment 500 bp PCR fragment 10 1kb PCR fragment 11 100 bp DNA Ladder 300 ng 12 Low DNA Mass Ladder 250 ng Agarose GP WO CON aA OFF WN rnm 4 single comb gel Results obtained using a 4 agarose gel are shown below using 20 uL per lane Lane Sample 50 bp DNA Ladder 300 ng 50 bp DNA Ladder 300 ng 450 bp PCR fragment 500 bp PCR fragment 25 bp DNA Ladder 400 ng 25 bp DNA Ladder 400 ng 10 bp DNA Ladder 740 ng 10 bp DNA Ladder 740 ng 450 bp PCR fragment 10 500 bp PCR fragment 11 50 bp DNA Ladder 300 ng 12 50 bp DNA Ladder 300 ng 4 Agarose HA 12346 6 TF 8 8 1017 12 WO CON OD OFF BP WN 2 34 Results with E Gel Double Comb Gels Introduction Results obtained using double comb E Gel gels are shown below All gels were photographed using a Kodak EDAS120 system You can also use a mini transillum
39. 0 bp DNA Ladder 900 ng 50 bp DNA Ladder 700 ng 100 bp DNA Ladder 900 ng pUC19 EcoR I cut 50 ng E Gel with SYBR Safe 10 pUC19 uncut 50 ng invitrogen 11 E Gel Low Range DNA Marker 350 ng 12 Low DNA Mass Ladder 470 ng 2 Agarose GP 123456789MN fF WON A FF FP WON B Results using E Gel with SYBR Safe Agarose Gels continued Examples using different imaging methods 2 Agarose GP 2 Agarose GP ae SS we em X Ww 12345 6789 MONRE 1234587382907 DNA samples run on an E Gel 2 with SYBR Safe are shown below recorded using different imaging methods Samples were loaded in a total volume of 20 uL and visualized using the indicated transilluminator and filter Photographs were taken using the MiniBis photo documentation system from DNR Standard 312 nm UV Standard 312 nm UV Blue light transilluminator transilluminator transilluminator Long pass ethidium SYBR Safe photographic SYBR Safe photographic bromide filter filter filter c M 2 Agarose GP RR a A M IM ese CS a 8080 M c E Gel with SYBR Safe E Gel with SYBR Safe E Gel with SYBR Safe invitrogen invitrogen amp invitrogen fo b Sample Low DNA Mass Ladder 470 ng E Gel Low Range DNA Marker 350 ng 240 bp PCR product 500 ng 317 bp PCR product 700 ng 1 kb PCR product 100 ng 100 bp DNA Ladder 900 ng 50 bp DNA Ladder 700 ng 100 bp DNA Ladder 900 n
40. 0 nm UV transilluminator Imaging systems such as laser based scanners equipped with an excitation source in the UV range or between 470 530 nm View SizeSelect agarose gels using amber filter or amber viewing goggles For imaging with a laser based scanner verify the system has an excitation source compatible with the proprietary dye Note If you plan to excise bands for cloning use a blue light transilluminator to visualize your DNA UV light sources in combination can lead to reduced cloning efficiencies Using a blue light transilluminator will also minimize your personal UV exposure Photograph E Gel SizeSelect agarose gels using a CCD camera or a laser based scanner For photographing gels refer to page 127 to determine the optimal filter sets to use or contact the instrument manufacturer for advice Do not use ethidium bromide filters that block light above 500 nm for photographing E Gel SizeSelect agarose gels E Gel SizeSelect agarose gels have greater sensitivity than ethidium bromide stained gels As a result a shorter exposure time or lower gain setting may be necessary Quantitation of DNA Isolated from E Gel SizeSelect Agarose Gels Quantitation Size and quantity of recovered DNA can be assessed by gel electrophoresis For accurate quantitation of DNA recovered from SizeSelect gels for applications such as next generation sequencing libraries we recommend performing qPCR For fluorometric quant
41. 1 8 sec Lane M 1 2 3 4 5 6 7 8 9 E Gel EX Agarose Gel 10 amp invitrogen Sample E Gel 1 Kb Plus DNA Ladder 5 kb PCR product 2 kb PCR product 1 kb PCR product 500 bp PCR product E Gel High Range DNA Marker 500 bp PCR product 1 kb PCR product 2 kb PCR product 5 kb PCR product E Gel 1 Kb Plus DNA Ladder An example of DNA samples run on an E Gel EX 2 agarose gel is shown below Samples were loaded in a total volume of 20 pL and visualized on the E Gel Safe Imager Real time Transilluminator Photographs were taken using the MiniBis photo documentation system from DNR and the SYBR Safe photographic filter using an exposure time of 1 8 sec Lane M 1 2 3 2 4 v 6 LE Rr I 7 SD 8 E Gel EX Agarose Gel 9 10 amp invitrogen Sample E Gel 1 Kb Plus DNA Ladder 500 bp PCR product 200 bp PCR product 100 bp PCR product 50 bp PCR product E Gel 1 Kb Plus DNA Ladder 50 bp PCR product 100 bp PCR product 200 bp PCR product 500 bp PCR product E Gel 1 Kb Plus DNA Ladder 55 Results using E Gel EX Agarose Gels for RNA Samples E Gel EX 1 agarose gel E Gel EX 2 agarose gel 56 An example of mouse brain RNA samples and the 0 1 2 Kb RNA Ladder see page 110 run on an E Gel EX 1 agarose gel is shown below Samples were loaded in a total volume of 20 uL and visualized on the E Gel Safe Imager Real time Transilluminator Photographs were taken usi
42. 100 ng well 4 5 8 9 20 21 40 41 PCR product 240 bp 100 ng well 6 7 17 18 30 31 41 42 PCR product 317 bp 100 ng well 8 9 16 17 28 29 44 45 PCR product 1 kb 100 ng well 10 11 14 15 26 27 45 47 PCR product 3 kb 100 ng well 12 13 36 37 E Gel 96 Low Range Quantitative Ladder 10 pL well 25 48 M upper left upper right 50 bp DNA Ladder 0 4 ug well Results with E Gel 48Gels Continued 4 E Gel 48 Gel Results obtained using a 4 E Gel 48 gel are shown below Electrophoresis was performed for 20 minutes using the standard conditions described in this manual and imaged using a KODAK EDAS290 system You can use a mini transilluminator to view the bands You may vary the amount of markers loaded on the gel to improve gel imaging 4 Agarose HR M 123 456 7 8 9 10111213141 1 io us dS 9 H ML ye i X TS The gel contains following samples Lane 46 47 M upper left 1 20 21 38 39 48 M lower right 24 25 M lower left 26 27 M upper right 6 13 14 34 35 2 3 6 7 4 5 22 23 60 bp 100 ng well 9 10 44 45 11 12 36 37 1920 29 16 30 31 17 92 29 18 40 41 19 42 43 Sa a UR e es ws ms n em 10 bp DNA Ladder 1 ug well 25 bp DNA Ladder 0 5 ug well 50 bp DNA Ladder 0 5 ug well 100 bp DNA Ladder 0 5 ug well E Gel Low Range Quantitative DNA Ladder 10 uL well Synthetic 21 mer siRNA short in
43. 127 to determine the optimal filter sets to use or contact the instrument manufacturer for advice Optimize settings of your system for E Gel CloneWell empirically You may need to increase the exposure time or gain setting Use IR blocking filter or emission filter with IR coating Use a blue light transilluminator such as the Safe Imager 2 0 Blue Light Transilluminator Cat no G6600 Use the REVERSE program of the iBase Power System to run the band backwards into the collection well see the iBase Power System manual Refill the second row with sterile water until the well is full prior to running your band of interest into the collection well Collect DNA from the well in two or more fractions Be sure to load the recommended DNA amount DNA Purification Using E Gel SizeSelect Agarose Gels Sample Preparation About E Gel SizeSelect agarose gels Materials needed DNA sample capacity Note Preparing samples Loading buffer E Gel SizeSelect 2 pre cast agarose gels feature two rows of wells a top row for loading samples and a bottom row to retrieve your DNA bands of interest The gels contain a proprietary fluorescent nucleic acid stain that is visualized by blue light transilluminator excitation emission at 490 522 nm and allows detection down to 1 5 ng band of DNA The recovered DNA has been shown to be compatible with nick translation and amplification steps without further purification
44. 27 to determine the optimal filter sets to use or contact the instrument manufacturer for advice Optimize settings of your system for E Gel EX agarose gels empirically You may need to increase the exposure time or gain setting Use IR blocking filter or emission filter with IR coating Use a blue light transilluminator such as the Safe Imager 2 0 Blue Light Transilluminator Cat no G6600 DNA Purification Using E Gel CloneWell Agarose Gels Sample preparation About E Gel CloneWell agarose gels Materials needed DNA sample capacity Preparing samples Loading buffer E Gel CloneWell pre cast 0 8 agarose gels provide a novel way to purify DNA bands with no purification necessary for downstream applications such as cloning E Gel CloneWell gels contain the safer and environmentally friendly SYBR Safe DNA gel stain enabling visualization of bands with a blue light transilluminator thus minimizing DNA damage For optimal results follow the guidelines for preparing your DNA sample as described in this section DNA sample Molecular weight markers page 60 Optional E Gel Sample Loading Buffer page 112 Refer to the following table to determine the amount of sample to run on an E Gel CloneWell agarose gel If you are unsure how much sample to use test a range of concentrations to determine the optimal concentration for your particular sample Note Exceeding the maximum amount of DNA resu
45. 30 minutes for single comb gels or 15 minutes for double comb gels Do not run longer than 45 minutes single comb gels or 25 minutes double comb gels Longer run times will damage the gel Do not allow the current to exceed 60 mA Turn down the voltage to decrease current At the end of the run remove the gel cassette from the power unit and analyze your results on a UV transilluminator For support visit www lifetechnologies com support or email techsupport alifetechn com www lifetechnologies com technologies August 2014
46. 6703 For more information contact Technical Support see page 110 The Caution symbol denotes a risk of safety hazard Refer to accompanying documentation The E Gel Safe Imager Real time Transilluminator is classified as a Class 1 LED product which is indicated by the symbol to the left A yellow label is affixed to the side of the E Gel Safe Imager Amber filter saying Caution Class 2 LED radiation when open do not stare into the beam Appendix C Two Step Loading Protocol Two Step Loading of E Gel Agarose Gels Introduction For optimal results follow the guidelines for preparing your DNA sample as described in this section Recommended The recommended total sample volume for each gel type is listed in the table below Volumes Note For best results keep all sample volumes uniform If you do not have enough samples to load all wells of the gel load an equal volume of deionized water all E Gel gels or buffer containing the same salt concentration as samples E Gel 48 96 gels into any empty wells Total Volume E Gel with SYBR Safe 10 uL 10 uL Gel Type Second Step E Gel single comb gel 10 pL 10 pL E Gel double comb gel 10 uL 10 pL E Gel EX agarose gel 10 pL 10 uL E Gel 48 gel 10 uL E Gel 96 gel 10 pL E Gel SizeSelect agarose gel 10 pL ENT Loading Buffer Loading buffer is required for the Two Step Loading method for E Gel agarose gels Mix the required amount of DNA with the loading buffe
47. 76 in a final sample volume of 10 uL see page 26 for details 118 Appendix D Opening E Gel Cassettes E Gel Opener E Gel Opener The E Gel Opener is an easy to use device specifically designed to open any E Gel single comb double comb or E Gel with SYBR Safe cassette for staining excision of DNA fragments or for blotting The E Gel Opener consists of an anodized aluminum platform housing two recessed steel blades one which is stationary and one which is movable The blades are brought into contact with the E Gel cassette by turning the large knob clockwise Blade Table edge i Do not use the E Gel Opener to open the E Gel 48 or 96 cassettes E Gel 48 and 96 RSSA cassettes are not designed to be opened E e Before using the E Gel Opener for the first time we recommend that you practice M M opening a few used E Gels to familiarize yourself with the process Practice on E xl E 7 Gels that will not be used further for preparative purposes e Electrophoresis must be complete before opening the E Gel We recommend that you place the E Gel on the transilluminator and photograph the gel before proceeding further If you plan to isolate DNA from the E Gel we recommend that you open the gel and excise the gel fragment immediately after electrophoresis as bands will diffuse within 20 minutes If you plan to blot the gel keep your blotting apparatus ready before opening the gel The blades on the E
48. A samples Non denaturing conditions Denaturing agents Denaturing conditions Important E Gel EX agarose gels can be used to run RNA samples RNA can be run under denaturing or non denaturing conditions Use non denaturing conditions only when checking for RNA quality where accurately determining size is not critical 1 Mix RNA sample with RNase free water such that the final volume is 20 pL 2 Do not heat Load the entire sample onto the E Gel EX 3 Run RNA using the E Gel EX EX 1 2 program program 7 for 10 minutes The only denaturing agent that is compatible with the E Gel EX system is Formamide 50 95 Lower concentrations are also acceptable There are two methods for denaturing your RNA sample to run on an E Gel EX agarose gel Method 1 1 Mix RNA 250 ng 2 ug sample with formamide to 50 95 such that the final volume is 20 uL Heat samples at 65 C for 5 minutes to denature RNA Place samples on ice immediately after heating Load entire sample onto E Gel EX g e V N Run RNA using the E Gel EX 1 2 program program 7 for 10 minutes Method 2 1 Mix RNA 250 ng 2 ug sample with RNAse free water or loading buffer such that the final volume is 20 uL 2 Heatsamples at 65 C for 5 minutes to denature RNA Using other denaturing agents like Glyoxal Formaldehyde or Urea results in very poor separation and band morphology on E Gel EX It is not recommended to run samples that we
49. Appendix F E GeL iBase Power Syst em naan ci nich pao ra dec Eo NV REI ERE a dee de aaaea 128 Downloading Firmware Upgrades for the E Gel iBase eessssssseeeeseseeeeerertetenetetetnentnenenentnene 128 Parameters for E Cel Base DIOSES eee eee ee ene eee ec ee ecce ts tat id federe Dedit edes 129 Appendix G E Gel PowerBase Version 4 esee esee crees eene nnne nnn n nemen anna nnn a suas annm assa aas n assa ass m rats 130 Usine the E Gel PowerDase Version d cias edes emt p ett a pedtebcnte oaa Reed eateries 130 Using E Gel NGS and E Gel with SYBR Safe Gels with the PowerBase v 4 sss 131 Appendix Hs E Gel Base seunsoocstistbiaitictudertiaieceuabin de cervo CE DX EE Ru CEDE EIE S CTI S E Eee Oria Eus 132 Using MEER er 37 gt ol MN IN 132 General Information Purpose of the guide Shipping and storage vi The E Gel Technical Guide contains information about E Gel pre cast agarose gels and is intended to supplement the Quick Reference Cards supplied with E Gel products Details for sample preparation and electrophoresis conditions are included in this guide To request the Quick Reference Card QRC or for additional information contact Technical Support page 110 or download the appropriate ORC from www lifetechnologies com All E Gel agarose gels are shipped at room temperature Store E Gel pre cast gels at room temperature Do not allow the temperature to drop below 4 C or rise abov
50. For optimal results follow the guidelines for preparing your DNA sample described in this section DNA sample Molecular weight markers page 49 Optional E Gel Sample Loading Buffer page 112 For optimal results refer to the following tables when determining the amount of sample to run on an E Gel SizeSelect agarose gel If you are unsure how much to use test a range of concentrations to determine the optimal concentration for your particular sample Excess DNA will cause poor resolution Note For best results keep all sample volumes uniform If you do not have enough samples to load all the wells of the gel load an identical volume of deionized water into any empty wells Single DNA Multiple DNA Optimal Sample Maximum Band Bands Amount Sample Amount 1 300 ng 1 100 ng band 5 150 ng 500 ng The proprietary fluorescent nucleic acid stain in E Gel SizeSelect agarose gels is more sensitive than ethidium bromide For downstream applications such as cloning or sequencing be sure to load enough DNA for your application as quantities of DNA that are sub optimal for your purposes can still produce a strong signal Prepare your samples as described below iF Prepare the samples by adding deionized water to the required amount of DNA to bring the total sample volume to 20 25 pL Prepare the DNA molecular weight marker by adding deionized water to the required amount of DNA to bring the total sample volume to 5 10 pL In
51. Imager 2 0 Blue Light E Gel Safe Imager Real Transilluminator time Transilluminator Viewing surface 19 x 19 cm 6 2 x 7 7 cm dimensions Overall dimensions 29 5 x 32 5 x 65 cm 20 0 x 11 0 x 4 3 cm Lamp life 50 000 hours 50 000 hours Included Amber filter unit and viewing Amber filter unit and viewing accessories glasses for viewing results glasses for viewing results Emission maxima 470 nm 480 nm Use the E Gel Safe Imager Amber filter unit or E Gel Safe Imager viewing glasses to help visualize SYBR Safe stained DNA and also prevent prolonged exposure to intense blue light Safe Imager Trans Emission spectra for the Safe Imager Blue Light illuminators are designed Transilluminator for viewing stained gels on the laboratory bench top and are compatible with E Gel with SYBR Safe gels E Gel EX gels E Gel CloneWell gels and E Gel SizeSelect gels Light from a LED source within the Safe Imager Blue Light Transilluminators passes through a blue filter producing a single Wavelength nm intensity signal at approximately 470 nm effective for the excitation of SYBR DNA binding dyes such as SYBR Safe DNA gel stain as well as many of our protein gel stains such as SYPRO Ruby SYPRO Orange and Pro Q Diamond stains Sensitivity obtained using this instrument is comparable to that obtained with a standard UV transilluminator t7 Safe Imager Blue Light Transilluminators Continued E Gel
52. Safe The E Gel Safe Imager Real time Transilluminator is designed for viewing E Gel with Imager Real time SYBR Safe E Gel CloneWell E Gel EX and E Gel SizeSelect gels on the laboratory i bench top for real time monitoring on the E Gel iBase Power System or for Transilluminator P 5 y ni documentation purposes at the end of the run directly on the E Gel Safe Imager E Gel Safe Imager Real time The E Gel Safe Imager Real time Transilluminator Transilluminator top has the following features e An array of 12 LED sources behind a blue filter that emit high intensity blue light e A red ON OFF button located at the front e 30 seconds and 5 minutes automatic shut off options A LED indicator light just behind the ON OFF 7 button to indicate the status of the Safe Imager E Gel Safe Imager Real time A short electrical cord to connect to the iBase USB port rene M d duc e USB port to enable future program updates Light Source LED Indicator light 4 ON OFF button Power inlet Attached short electrical cord E Gel Safe Light from the array of 12 Emission spectrum for the E Gel Safe Imager i LED sources within the Real time Transilluminator mager E Gel Safe Imager Real Real time time Transilluminators passes Transilluminator through a blue filter producing a single intensity signal at approximately 480 nm effective for the excitation of SYBR
53. UK versions PIE secas 5 G6500STUK Safe Imager 2 0 Blue Light Transilluminator 1 G6600 Safe Imager Viewing Glasses 1 537103 Safe Imager International Power Cord replacement 1 537104 Safe Imager Amber Filter Unit replacement 1 537105 Loading buffers are optional for E Gel agarose gels The following loading buffers are suitable for use with E Gel agarose gels but should be diluted 50 200 fold before use Product Quantity Catalog no 10X BlueJuice Gel Loading Buffer 10x 3x1mL 10816 015 TrackIt Cyan Orange Loading Buffer 3x0 5mL 10482 028 TrackIt Cyan Yellow Loading Buffer 3 x 0 5 mL 10482 035 Technical Support Obtaining support For the latest services and support information for all locations go to www lifetechnologies com At the website you can e Access worldwide telephone and fax numbers to contact Technical Support and Sales facilities e Search through frequently asked questions FAQs e Submit a question directly to Technical Support techsupport lifetech com e Search for user documents SDSs vector maps and sequences application notes formulations handbooks certificates of analysis citations and other product support documents e Obtain information about customer training e Download software updates and patches Safety Data Sheets Safety Data Sheets SD5s are available at www lifetechnologies com support SDS Certificate of The Certificate of Analysis provides detailed quality control and product q
54. USER GUIDE E Gel Technical Guide General information and protocols for using E Gel pre cast agarose gels Publication Part Number MANO0000375 Revision A 0 For Research Use Only Not for use in diagnostic procedures d technologies For Research Use Only Not for use in diagnostic procedures Information in this document is subject to change without notice DISCLAIMER LIFE TECHNOLOGIES CORPORATION AND OR ITS AFFILIATE S DISCLAIM ALL WARRANTIES WITH RESPECT TO THIS DOCUMENT EXPRESSED OR IMPLIED INCLUDING BUT NOT LIMITED TO THOSE OF MERCHANTABILITY FITNESS FORA PARTICULAR PURPOSE OR NON INFRINGEMENT TO THE EXTENT ALLOWED BY LAW IN NO EVENT SHALL LIFE TECHNOLOGIES AND OR ITS AFFILIATE S BE LIABLE WHETHER IN CONTRACT TORT WARRANTY OR UNDER ANY STATUTE OR ON ANY OTHER BASIS FOR SPECIAL INCIDENTAL INDIRECT PUNITIVE MULTIPLE OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT INCLUDING BUT NOT LIMITED TO THE USE THEREOF IMPORTANT LICENSING INFORMATION This product may be covered by one or more Limited Use Label Licenses By use of this product you accept the terms and conditions of all applicable Limited Use Label Licenses TRADEMARKS 2014 Thermo Fisher Scientific Inc All rights reserved All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified ii Table of Contents Generab bal 6 alag te a 0 a ec tn ere racers c eee ron ter
55. Up Down A V buttons to change the program Note The SPEED E Gel program is available for quick runs see page 79 e Go Jn 3 Set time to Run Time to Reference Line according to the Run Time Estimation Table see page 76 for the band size of the DNA fragment to be collected Default run time for Run SizeSelect 2 is 8 minutes To change the run time press the Mode button until the minutes or seconds blink and change the values using the Up Down buttons up to the maximal run time of 20 minutes A Moa o y 4 Run your band of interest to the reference line Monitor the run periodically and press the Go button to stop the run when the band reaches the reference line Reference Line Reference Line NE i 3 Bands reaching the inp eji Line The device stops automatically when the programmed time has elapsed A flashing red light and beeping rapid beeping for 30 seconds followed by a single beep every minute signals the end of the run Press and release the Go button to stop the beeping The LED shows a steady red light If the band has not reached the reference line run the gel until the band reaches the line 77 Collecting DNA Using E Gel SizeSelect Agarose Gels Continued Electrophoresis Using iBase Power System continued 78 When the band reaches the reference line refill the collection wells to 25 uL with sterile water The refill volume may vary between wells Do not overfill
56. Use fresh gel cassette Use properly stored gels before the cassette specified expiration date E Gel EX cassette is not Remove cassette and reinsert a steady red light illuminates inserted properly into a base on the base when the cassette is correctly inserted and power is on Incorrect adaptor used Use only UL Listed Class 2 Direct Plug in Adaptor included with the E Gel iBase Poor resolution or Sample is overloaded Do not load more than the recommended amount of DNA smearing of bands sample per band see page 48 High salt concentration Dilute your high salt samples as described on page 26 Aberrant pre run step Do not pre run E Gel EX agarose gels Very low volume of sample Avoid introducing bubbles while loading the samples loaded or sample was not Bubbles will cause band distortion loaded properly Load the recommended sample volume based on the gel type and loading method For proper band separation we recommend keeping sample volumes uniform Load deionized water into any empty wells Gel was not electrophoresed For best results run the gel within 1 minute of sample immediately after sample loading loading Expired gel used Use properly stored gels before the expiration date Longer electrophoresis run Longer run times cause an increase in the current resulting time or high current during in poor band migration or a melted gel Do not run the gel the run longer than recommended time for each gel type
57. a or a laser based scanner e For photographing gels refer to page 127 to determine the optimal filter sets to use or contact the instrument manufacturer for advice Do not use ethidium bromide filters that block light above 500 nm for photographing E Gel CloneWell agarose gels While yielding similar sensitivities to ethidium bromide SYBR Safe is somewhat dimmer yet with a lower background than ethidium bromide As a result a slightly longer exposure time or higher gain setting may be necessary 67 Results using E Gel CloneWell Agarose Gels Introduction On this page we display typical results using E Gel CloneWell agarose gels Example of E Gel An example of DNA samples run on an E Gel CloneWell gels is shown below Bands CloneWellL that have entered the Collection Well completely are indicated by arrows The gel was visualized on a Safe Imager transilluminator Collection Wells Bands that have entered the Collection Well CloneWell E Gel CloneWell gels were tested in cloning experiments using restriction based cloning TOPO cloning and Gateway cloning In all cases colony counts were several fold higher using E Gel CloneWell compared to ethidium bromide gels Exact numbers may vary depending on the experiment but you should get many more colonies when using E Gel CloneWell for your fragment isolation cloning 68 Troubleshooting No current Copper contacts in the base Mak
58. adder 10597 011 50 bp DNA Ladder 10416 014 28 Molecular Weight Markers Continued Double comb E Gel gels E Gel 1 Kb Plus DNA Ladder 10488 090 Load 100 250 ng E Gel High Range DNA Marker 12352 019 Markers in a volume of 0 8 10 uL in marker well Low DNA Mass Ladder 10068 013 High DNA Mass Ladder 10496 016 E Gel 1 Kb Plus DNA Ladder 10488 090 3 E Gel Low Range Quantitative DNA Marker 12373 031 TrackIt 50 bp DNA Ladder 10488 043 TrackIt 1 Kb Plus DNA Ladder 10488 085 29 Using E Gel Agarose Gels with the iBase Power System Introduction After preparing your samples proceed with electrophoresis Instructions are provided below to load and run E Gel single comb and double comb gels using the E Gel iBase Power System For details on using E Gel agarose gels with the E Gel PowerBase v 4 see page 130 For details on using E Gel agarose gels with the E Gel Base see page 132 Insert a cassette in 1 Attach the power cord of the iBase device to the power inlet and then to the electrical the iBase Power outlet Use only properly grounded AC outlets and cords System 2 Open the package and remove the gel Do not remove the comb until you start loading the samples 3 Slide the cassette into the two electrode connections on the iBase device Press down on the left side of the cassette to secure it into the iBase device The two electrodes on the right side of the gel cassette must be in contact
59. am your robotic loading system to set the A1 tip of the 8 12 or 96 tip robotic head over the E Gel 96 gel cassette as described below Set the position of the first tip approximately 1 mm above the slope of the A1 well see Figure 2 below This will ensure that the remaining tips are aligned above the slopes of the remaining wells Refer to the manufacturer s manual of your robot to program this setting After programming the setting load your samples During loading the samples will fall onto the slopes of the wells and be drawn into the wells by capillary force Figure 1 12 3 4 5 6 7 8 9 1011 12 M A fO L3 O L3 L3 CHC da ad CHO B rj pcm gg bh EN E L C L3 L3 T3 D3LDL3 a aa a aag aa a oo D LL pE E hh OC O DO OLC CO CO EA OAA AA FAO a AO 0 O GAO F O E EK O O OCO O DO O O O C G CH L3 L3 L3 CH CACHE CACHE OA AOI CHO H I p Ep bp D OCOC bh HB EJ E Figure 2 ae E Pd E Loading E Gel 48and 96 Gels Using the Barcode Each E Gel 48 and 96 gel is labeled with an individual barcode with a number The barcode facilitates identification of each gel cassette during the electrophoresis of multiple gels Each E Gel 48 and 96 gel contains an EAN 39 type of barcode which is recognized by the majority of commercially available robotic barcode readers Refer to the manufacturer s instructions to set up the barcode reader Note When capturing an image of the E Gel 48 or 96 gel note that the barcode label is easily o
60. around the unit to prevent overheating Plug the power cord into electrical outlet 2 Before handling your gel or sample ensure that the personal safety equipment you are using is appropriate for the hazards posed by the chemicals that may be present Place the gel or sample onto the surface of the Safe Imager transilluminator 3 Place the amber filter unit on top of the sample or stained gel If you are using a gel that is larger than the viewing area you may rest the amber filter unit directly on top of the gel or use the viewing glasses instead The viewing glasses are useful when excising bands from DNA gels as they allow the bands to be visualized while leaving the gel surface unobstructed 4 Switch the Safe Imager transilluminator ON using the ON OFF switch located at the front of the instrument Any SYBR stained DNA present in solution or in gel bands should be immediately visible after the light is on and the amber filter unit or viewing glasses are in position Safe Imager Blue Light Transilluminators Continued Imaging Cleaning and maintenance e To document your results you may use any standard imaging device Due to the small footprint the Safe Imager transilluminators may fit inside the cabinet of your current gel documentation system In many cases satisfactory results are obtained by placing the amber filter unit on top of the gel and photographing imaging using standard procedures e Your CCD documentati
61. atically shuts off The flashing green light changes to a flashing red light and the PowerBase beeps rapidly 5 Press and release either button to stop the beeping you will hear only one beep The light will change from a flashing red light to a steady red light 130 Using E Gel NGS and E Gel with SYBR Safe Gels with the PowerBase v 4 Electrophoresis 1 Choose the appropriate run time for your gel on the E Gel PowerBase v 4 using PowerBase v4 Gal Ty For the 30 minute run press and release the 30 min button to start electrophoresis A steady green light indicates the start of the 30 minute run For the 15 minute run press and release the 15 min button to start electrophoresis A steady blue light indicates the start of the 15 minute run Note The actual running time of the E Gel gel may vary between 15 17 minutes for double comb gels and 30 33 minutes for single comb and E Gel with SYBR Safe gels 2 The current automatically shuts off at the end of each run The E Gel PowerBase v 4 signals the end of the run with a flashing red light and rapid beeping 3 Press and release either button to stop the beeping A steady red light indicates that the E Gel PowerBase v 4 is in Ready Mode 4 Atthe end of the run remove the gel cassette from the power unit and analyze your results 131 Appendix H E Gel Base Using the E Gel Base Introduction Pre run with an E Gel Base Running E Gel
62. bands for cloning use a blue light transilluminator to visualize your DNA UV light sources can lead to reduced cloning efficiencies Using a blue light transilluminator will also minimize your personal UV exposure e Photograph E Gel EX agarose gels using a CCD camera or a laser based scanner e For photographing gels refer to page 127 to determine the optimal filter sets to use or contact the instrument manufacturer for advice Do not use ethidium bromide filters that block light above 500 nm for photographing E Gel EX agarose gels E Gel EX agarose gels have greater sensitivity than ethidium bromide stained gels As a result a shorter exposure time or lower gain setting may be necessary E Gel EX agarose gels should be disposed of as hazardous waste in the same manner as ethidium bromide containing gels Contact your safety office or local municipality for appropriate disposal in your community Results using E Gel EX Agarose Gels Introduction E Gel EX 1 agarose gel E Gel EX 2 agarose gel On this page we display typical results using E Gel EX agarose gels 1 and 2 An example of DNA samples run on an E Gel EX 1 agarose gel is shown below Samples were loaded in a total volume of 20 pL and visualized on the E Gel Safe Imager Real time Transilluminator Photographs were taken using the MiniBis photo documentation system from DNR and the SYBR Safe photographic filter using an exposure time of
63. barcode readers page 87 The separation range for E Gel 48 gels is listed in the following table Sample Range bp Separation 1 E Gel 48 400 bp 600 bp 90 bp 600 bp 1 kb 100 bp 1 kb 4 kb 900 bp 4 kb 10 kb 1 kb 2 E Gel 48 100 bp 300 bp 29 bp 300 bp 700 bp 90 bp 700 bp 1200 bp 100 bp 1200 bp 2000 bp 200 bp 4 E Gel 48 9 bp 40 bp 9 bp 40 bp 80 bp 10 bp 80 bp 175 bp 20 bp 175 bp 300 bp 90 bp 300 bp 600 bp 100 bp High Throughput E Gel Electrophoresis System High throughput The system consists of the following components E Gel e electrophoresis system E Gel 96 gels Each E Gel 96 gel contains 96 sample lanes and 8 marker lanes in a patented staggered well format and is designed for high throughput agarose electrophoresis of nucleic acids E Base Electrophoresis Device The E Base is a base and a power supply all in one device and is an easy to use pre programmed device specifically designed for electrophoresis of E Gel 48 and 96 gels E Holder Platform The E Holder Platform is designed to hold E Gel 96 gels during robotic loading The E Holder is used to load multiple gels on a robotic platform while other gels are running on the E Base E Editor 2 02 Software The E Editor 2 02 software allows you to quickly reconfigure digital images of E Gel 48 or 96 gel results for analysis and documentation The E Editor 2 02 software can be downloaded for free from our Website at www li
64. ble comb gel are compatible for loading with a multichannel pipettor Each E Gel 48 gel contains 48 sample wells and 4 marker wells M Cassette Size Gel Thickness Gel Volume Gel Percentage Well Depth Dimensions of the Well Running Distance one well to the next Space between Well Center 13 5 cm 1 x 10 8 cm w x 0 67 cm thick 3 mm 50 mL 196 2 and 4 3 mm 3 6 mm l x 2 2 mm w 32 mm 4 5 mm The wells of the E Gel 48 gel are compatible for loading with a multichannel pipettor Each E Gel 96 gel contains 96 sample wells and 8 marker wells M Cassette Size Gel Thickness Gel Volume Gel Percentage Well Depth Well Opening Well Bottom Running Distance one well to the next Space between Wells 13 5 cm 1 x 10 8 cm w x 0 67 cm thick 3 mm 50 mL 1 and 2 3 mm 3 8 mm x 1 8 mm 3 3 mm x 1 1 mm 16 mm 9 mm The wells of the E Gel 96 cassette are compatible with a multichannel pipettor or 8 12 or 96 tip robotic loading devices Product Specifications Continued E Base specifications Dimensions Weight Safety Temperature Built in Features LED The specifications for Mother E Base and Daughter E Base are listed below 14 6 cm x 15 cm x 5 3 cm Mother E Base 370g Daughter E Base 271g Double Insulation UL listed and CE certified Ambient 15 C to 40 C Digital time display 00 99 minutes alarm light The SBS Society
65. bp DNA Ladder TrackIt 100 bp DNA Ladder 50 bp DNA Ladder 100 bp DNA Ladder Low DNA Mass Ladder E Gel Low Range Quantitative DNA Marker TracklIt 25 bp DNA Ladder TrackIt 50 bp DNA Ladder 25 bp DNA Ladder 50 bp DNA Ladder E Gel Low Range Quantitative DNA Ladder E Gel 96 gels 1 2 E Gel Low Range Quantitative DNA Ladder 10787 018 10488 085 10594 018 12352 019 10488 043 10488 058 10416 014 15628 019 10068 013 12373 031 10488 022 10488 043 10597 011 10416 014 12373 031 E Gel 96 High Range DNA Marker 12352 019 12373 031 Amount Used Load 100 250 ng of markers in a volume of 15 uL in marker well Use a buffer containing the same salt concentration as your samples Load 100 250 ng of markers in a volume of 20 uL in marker well Use a buffer containing the same salt concentration as your samples 65 General Guidelines for E Gel 48 96 Gels Continued Robotic Platforms Aligning the Robotic Loading Assembly 86 The Mother E Base and Daughter E Base are designed to fit most robotic platforms allowing you to load and run E Gel 48 and 96 Gels directly on the robot If you need to load multiple gels on a robotic platform while other gels are running on the E Base use an E Holder Platform page 95 for details The wells of the E Gel 96 gel are staggered to provide maximum run length see Figure 1 below For proper loading of samples it is important to progr
66. cted to an electrical outlet E Gel 96 cassette inserted into a base Press and release the power button Automatic Automatic Press and release the power button during the run Press and release the power button Continuous beeping for 2 minutes followed by a single beep every minute Continuous beeping for 2 minutes followed by a single beep every minute No light if a cassette is not inserted or red light if a cassette is inserted Steady red Steady green Flashing red until the time button is pressed Alternating red and green With gel cassette in steady red Without gel cassette no light Steady red Default time setting 12 minutes or last time setting Count down time Negative time display 00 to 19 minutes Negative time display 00 to 19 minutes Flashing time display Last time setting E Gel 96 Mother Daughter Base Quick Reference Guide Continued Mode Action Sound Light Digital Display Restart after Press and Steady green Count down a manual release the time stop power button Return to Press and hold With gel cassette Last time Ready mode the power in steady red setting after a button Without gel manual stop cassette no light Failure Remove the Rapid Steady red Flashing gel cassette beeping from the base No cassette Last time setting Time setting Press and release the increases by time button 1 minute
67. ctions on using the gels with E Gel 96 mother base and daughter base see page 106 DNA sample Molecular weight markers page 85 Optional E Gel Sample Loading Buffer page 112 Use 20 100 ng DNA per band for samples containing one unique band or up to 500 ng per lane for samples containing multiple bands If you are unsure how much to use test a range of concentrations to determine the optimal concentration for your particular sample Excess DNA will cause poor resolution Single DNA Band Multiple DNA Bands 20 100 ng 500 ng Preparing Samples Prepare your samples based on the loading method used as described below Loading Buffer 64 E Gel 48 gel E Gel 96 gel 20 pL Add deionized water to the required Add deionized water to the required amount of DNA to bring the total amount of DNA to bring the total sample volume to 15 pL sample volume to 20 pL Loading buffer is optional See page 25 for more details General Guidelines for E Gel 48 96 Gels Continued DNA Molecular Weight Markers We recommend using the following DNA molecular weight markers for different types of E Gel agarose gels to obtain good resolution Note Supercoiled DNA molecular weight markers may produce a slightly fuzzy pattern when run on E Gel agarose gels containing ethidium bromide E Gel 48 gels 1 2 4 1 Kb Plus DNA Ladder TrackIt 1 Kb Plus DNA Ladder 500 bp DNA Ladder E Gel High Range DNA Marker TrackIt 50
68. ctrode connections on the base The LED produces a steady red light to indicate that the cassette is correctly inserted Slide cassette into electrodes Press left side to secure 4 Remove the combs and load your samples Be sure to load molecular weight markers and add water to any empty wells Note It is not necessary to pre run E Gel CloneWell agarose gels Load the E Gel Load the E Gel CloneWell agarose gel within 15 minutes of opening the pouch and run agarose gel the gel within 1 minute of loading samples Avoid introducing bubbles while loading as bubbles will cause bands to distort 1 Remove the comb from the E Gel CloneWell gel using both hands to lift the comb gently by rolling the comb slowly towards you Be careful to pull the comb straight up from both sides Do not bend the comb Remove any excess fluid using a pipette Load 20 25 uL prepared sample per sample well in the top row see page 59 for details 3 Load 5 10 pL of the appropriate molecular weight marker page 60 in the small middle well 4 Load 25 pL of water into any remaining empty wells of the top row Load 25 30 uL of water into the wells of the bottom row Before loading the gel make sure you have a blue light transilluminator set up for operant viewing the bands See page 61 for instructions on setting up the blue light transilluminator and page 67 for details on viewing bands 62 Collecting DNA Using E Gel CloneWell Agaro
69. current during in poor band migration or a melted gel Do not run the gel the run longer than recommended time for each gel type Melted gel Increased current due to Do not run the gel longer than 40 minutes longer run times Sample leaking from Sample is overloaded Load the recommended sample volume per well the wells Wells damaged during comb Remove the comb gently without damaging the wells removal 69 Troubleshooting Continued Observation Failure Mode indicated by a flashing red light and continuous rapid beeping Speckles visible High background suboptimal or no image Stripes visible on image Low cloning efficiency Band of interest below collection well Low volume for collection Low yield 70 Cause Defective cassette Cold cassette or improper operating conditions Dust fluorescing in same wavelength as SYBR Safe No filters or wrong filter set Photographic settings not optimal No IR coating on camera Used a UV light source to visualize DNA Run time too long Missed refilling water Band is too big Solution Disconnect the base and replace gel cassette with a fresh gel cassette Press and release the power button to return to Ready Mode Use a cassette stored at room temperature Avoid storing gel cassettes at 4 C Use E Gel iBase at room temperature 20 25 C Make sure gel is clean before imaging Refer to page
70. details 31 Using Single Comb and Double Comb Gels with the iBase Power System Continued Speed run using the iBase Power System Interrupt a run on the iBase Power System 32 The iBase device is pre programmed with a SPEED E Gel program for performing runs using high power to generate rapid yes no results The program is suitable for 0 8 1 2 and 2 E Gels only This program is limited to 7 minutes where the bands migrate less than half the length of the gel A run exceeding 7 minutes under these conditions results in a defective run This mode is not compatible with E Gel 4 gels Electrophoresis can be interrupted at any time by pressing and releasing the Go button to stop the current A flashing red light indicates that the current is stopped and the digital display flashes the message Press GO to Run Hold Go to Reset to indicate that the run was interrupted You can remove the gel from the iBase device to check the progress of the run then e Continue the run from the point at which it was stopped Reinsert the gel and press and release the Go button The light changes to a steady green and the LCD display shows the count down time The run time but not the program can be adjusted before continuing the run e Cancel the interrupted run Press and hold the Go button for a few seconds The LCD display resets and returns to Ready Mode A new program and run time can be selected to rerun the gel Result
71. dy computer to download firmware upgrades from www lifetechnologies com see page 128 E Gel iBase Power System top view To Electrical Outlet LCD Display i Down _ __ i amp button button E Gel iBase Power System back view i el il Ul Power USB inlet port E Gel iBase Power System Continued iBase and Safe Imager integrated system E Gel Safe Imager Real time Transilluminator E Gel iBase Power System and E Gel Safe Imager Real time Transilluminator form an integrated system for running and viewing SYBR Safe stained E gels The iBase fits neatly on the Real time Transilluminator and power is provided through a shared power cord adapter included with the E Gel iBase Power System With the matching amber filter mounted on top of the iBase included with the E Gel Safe Imager Real time Transilluminator you can follow the migration of DNA bands while they are running or document your results at the end of the run directly iBase and Safe Imager Integrated System E Gel iBase gt Power System Amber Filter E Gel PowerBase E Gel The E Gel PowerBase Version 4 figure below is an easy to use automated device PowerBase specifically designed to simplify electrophoresis of single comb or double comb E Gel agarose gels The E Gel PowerBase is a base and a power supply all in one device The operation of the E Gel PowerBase
72. e devices To honor the warranty E Base can only be opened and serviced by Life Technologies The protection provided by the equipment may be impaired if the equipment is used in a manner not specified by Life Technologies Life Technologies Israel Ltd a Life Technologies company is the manufacturer and owner of the UL file For more information contact Life Technologies Israel Ltd 12 Hamada St Rehovot Israel 76703 For more information contact Technical Support see page 110 The Caution symbol denotes a risk of safety hazard Refer to accompanying documentation WEEE Waste Electrical and Electronic Equipment symbol Class II product Explanation of Symbols and Warnings Continued E Gel iBase Power System CE LISTED E189045 FC E Gel PowerBase CC Du The E Gel iBase Power System complies with the Underwriters Laboratories Inc regulation and the European Community Safety requirements Operation of the E Gel iBase Power System is subject to the following conditions Indoor use Altitude below 2 000 meters Temperature range 5 to 40 C Maximum relative humidity 80 Installation categories over voltage categories II Pollution degree 2 Mains plug is a disconnect device and must be easily accessible Do not attempt to open the iBase Device To honor the warranty iBase device can only be opened and serviced by Life Technologies The protection provided by the equipment
73. e 40 C Gels are guaranteed to be stable for at least 2 to 6 months upon receipt e Standard and Clear gels are stable for at least 6 months e E Gel EX and E Gel SizeSelect are stable for at least 3 months e E Gel with SYBR Safe are stable for at least 2 months Please refer to the expiration date printed on the packaging of your E Gel agarose gel All electrophoresis bases are shipped at room temperature Store the E Gel Base E Gel iBase E Gel PowerBase and E Base at room temperature Avoid storing or using any electrophoresis bases at 4 C e Some E Gel agarose gels contain ethidium bromide a known mutagen The concentration of ethidium bromide in each gel ranges from 0 1 to 0 3 ug mL All E Gel agarose gels contain 0 055 Proclin added as a preservative apart from E Gel 48 4 gels which contain 0 01 Thimerosal Each gel is provided in a sealed package so you are protected from exposure As a precaution always wear gloves and protective clothing when handling the gel e Dispose of used E Gel agarose gels containing ethidium bromide E Gel EX and E Gel SizeSelect agarose gels as hazardous waste e Avoid overexposure of skin and eyes when using UV light e Avoid overexposure of eyes when using intense blue light e Avoid touching the gel during electrophoresis Introduction E Gel Electrophoresis System Introduction Advantages of E Gel agarose gels Throughput capacity The E Gel a
74. e bands You may vary the amount of markers loaded on the gel to improve gel imaging 1 Agarose GP Mi2aa 7 amp 1011 1212 14 15 16 17 10 19 20 21 22 29 24 M 22 d A L L pr iB e M Spis BI 11118 IKE NI EUIS The gel contains following samples Lane Sample 1 24 M lower left lower right High Mass DNA Ladder 4 uL well 2 3 22 23 32 33 36 37 40 41 PCR product 317 bp 100 ng well 4 5 20 21 30 31 42 43 PCR product 1 kb 100 ng well 6 7 18 19 28 29 A4 45 PCR product 3 kb 100 ng well 8 9 12 13 16 17 26 27 46 47 PCR product 9 kb 100 ng well 10 11 14 15 34 35 38 39 E Gel High Range DNA Ladder 10 uL well 25 48 M upper right upper left 1 Kb Plus DNA Ladder 0 5 ug well 97 Results with E Gel 48 Gels Continued 2 E Gel 48 Gel 98 Results obtained using a 2 E Gel 48 gel are shown below Electrophoresis was performed for 20 minutes using the standard conditions described in this manual and imaged using a KODAK EDAS290 system You can use a mini transilluminator to view the bands You may vary the amount of markers loaded on the gel to improve gel imaging 2 Agarose GP p M123 45 6 7 6 9 1011 12 13 1415 16 17 18 19 20 21 22 23 24 M The gel contains following samples Lane Sample 1 24 M lower left lower right 100 bp DNA Ladder 0 4 pg well 2 3 22 23 34 35 38 39 PCR product 150 bp
75. e only the power cord supplied with the Safe Imager transilluminator to power the device Attach the supplied power cord to the back of the Safe Imager transilluminator Plug the other end of the power cord into a properly grounded electrical outlet with the correct plug adaptor attached Always disconnect the Safe Imager transilluminator from the electrical outlet before cleaning the device Do not leave the Safe Imager switched on for extended periods of time After viewing and documenting the gel or sample always switch the unit off Do not attempt to open the Safe Imager The Safe Imager 2 0 Blue Light Transilluminator does not produce UV light but it does emit an intense blue light which may promote macular degeneration upon prolonged exposure especially in those prone to such problems e g people with fair complexion and blue eyes nutritional or endocrine defects or those who are aging Use the Safe Imager amber filter unit or Safe Imager viewing glasses provided with this device to protect your eyes The amber filter unit and viewing glasses are for viewing stained gels using the Safe Imager 2 0 Blue Light Transilluminator The amber filter unit will NOT protect your eyes when viewing gels on UV transilluminators and although the viewing glasses do block UV light they are not designed for use as UV safety glasses 1 Place the Safe Imager 2 0 Blue Light Transilluminator on a level surface with enough air circulation
76. e sure the copper contacts in the base are intact are damaged due to improper use Expired or defective gel Use fresh gel cassette Use properly stored gels before the cassette specified expiration date E Gel CloneWell cassette is Remove cassette and reinsert a steady red light illuminates not inserted properly intoa on the base when the cassette is correctly inserted and base power is on Incorrect adaptor used Use only UL Listed Class 2 Direct Plug in Adaptor included with the E Gel iBase Power System Poor resolution or Sample is overloaded Do not load more than 200 ng of sample DNA per band smearing of bands High salt concentration Dilute your high salt samples as described on page 26 Aberrant pre run step Be sure to pre run the gel but do not exceed 2 minutes Very low volume of sample Avoid introducing bubbles while loading the samples loaded or sample was not Bubbles will cause band distortion loaded properly Load the recommended sample volume based on the gel type and loading method For proper band separation we recommend keeping sample volumes uniform Load deionized water or TE into any empty wells Gel was not electrophoresed For best results run the gel within 15 minutes of sample immediately after sample loading loading Expired gel used Use properly stored gels before the expiration date Longer electrophoresis run Longer run times cause an increase in the current resulting time or high
77. ecommend using the following DNA molecular weight markers for different types of E Gel EX agarose gels to obtain good resolution Note Using DNA ladders with EDTA concentrations of gt 0 25 mM can result in low resolution and limited separation Also supercoiled DNA molecular weight markers may produce a slightly fuzzy pattern when run on E Gel EX agarose gels E Gel 1 Kb Plus DNA Ladder 10488 090 Load 100 250 ng E Gel High Range DNA Marker 12352 019 Markers ina volume of 20 uL High DNA Mass Ladder 10496 016 E Gel 1 Kb Plus DNA Ladder 10488 090 E Gel Low Range Quantitative DNA Marker 12373 031 Low DNA Mass Ladder 10068 013 E Gel 25bp DNA Ladder 10488 095 E Gel 50bp DNA Ladder 10488 099 49 Using E Gel EX Agarose Gels with the iBase Power System Introduction After preparing your samples proceed with electrophoresis Instructions are provided below to load and run DNA samples on E Gel EX gels using the E Gel iBase Power System For details on running RNA samples on E Gel EX gels see page 52 Install the iBase If using only the E Gel iBase Power System attach the power cord with the transformer to the power inlet of the iBase device and plug the other end of the power cord into an Power System l electrical outlet Use only properly grounded AC outlets and cords Install the iBase If using the E Gel iBase Power System in conjunction with the Safe Imager Real time Power System wi
78. ectrophoresis button to stop the current The stopped current is indicated by a steady red light and the Run digital display will flash to indicate that the run has been interrupted You can remove the gel from the mother or daughter base to check the progress of the run Then To continue the run from the point at which it was stopped reinsert the gel and press and release the power button To cancel the rest of the interrupted run press and hold the power button for a few seconds The digital display will reset and the base will return to ready mode If desired you can then program a new run time In case of a power failure the run will continue when the power resumes The mother or daughter base will signal the end of the run as described on the previous page except the light will be an alternating red green and ER is displayed in the digital display to indicate that a power failure has occurred during the run 107 E Gel 96 Mother Daughter Base Quick Reference Guide A quick reference guide for operating the E Gel 96 mother and daughter base is provided below Operating modes and electrophoresis runs are described below Mode Action Sound Light Digital ee Introduction 108 Base plugged in Ready with no current flowing through gel End of run Run ends after a power failure during the run Pause manually end the run Return to Ready mode after an automatic stop Mother base conne
79. en throughout the run e To turn on the light for 5 minutes press and hold the ON OFF button for a few seconds The LED indicator light will turn a steady green followed by a flashing green the last 30 seconds of the run Any SYBR Safe stained DNA present should be immediately visible after light is on and amber filter unit or viewing glasses are in position 3 To turn off the light press and release the ON OFF button The LED indicator light will turn red Note A flashing red LED indicates an error Wait until the LED turns a steady red before turning on the device again If the LED does not turn red after the run disconnect the Safe Imager and try again after a few minutes If this problem persists contact Technical Support page 110 Imager Real time Transilluminator 19 Safe Imager Blue Light Transilluminators Continued Safety Information Safe Imager 2 0 Blue Light Transilluminator Operating the Safe Imager Blue Light Transilluminator 20 The Safe Imager 2 0 Blue Light Transilluminator is an electrical device Never touch the power cord or outlet with wet hands Do not use this device in damp areas or while standing on damp floors The Safe Imager 2 0 Blue Light Transilluminator is supplied with an international power cord This power cord has a universal transformer compatible with either 110 V or 220 V electrical outlets and a selection of plug adaptors allowing use with any electricity supply Us
80. er manual 103 Troubleshooting Troubleshooting The table below provides some solutions to possible problems you might encounter during the electrophoresis of E Gel 48 and 96 agarose gels To troubleshoot problems with single and double comb E Gel see page 36 No current Daughter E Base used Do not use the Daughter E Base without a Mother without Mother E Base E Base The Daughter E Base does not have an electrical plug to connect to an electrical outlet Copper contacts in the base Make sure the copper contacts in the base are intact are damaged due to improper use Expired or defective gel Use fresh gel cassette Use properly stored gels before the cassette used specified expiration date Gel cassette is not inserted Remove cassette and reinsert a red light illuminates at the properly into a base lower left corner of the base a fan in the base begins to run and digital display indicates time for a selected program or last time setting Ready Mode when the gel is properly inserted into the base Poor resolution or Sample is overloaded Do not load more than 20 100 ng of sample DNA per band smearing of bands Less DNA is required since E Gel agarose gels are thinner High salt concentration Dilute your high salt samples as described on page 26 Very low volumes of sample Avoid introducing bubbles while loading the samples loaded or sample was not Bubbles will cause band distortion loaded properly Load
81. es Continued DNA ladders E Gel 1 Kb Plus Ladder Tracklt Ladders High salt samples 26 DNA ladders can be used to estimate the size of fragments and to track the progress of a run Suggested ladders are listed in the description for running each type of gel The E Gel 1Kb Plus DNA Ladder is recommended for use with E Gel EX and E Gel SizeSelect gels as well as other E Gel precast gels If using TrackIt DNA ladders for molecular weight estimation do not use more than 2 uL in a total load volume of 20 uL TrackIt DNA ladders are not recommended for use with E Gel EX or E Gel SizeSelect agarose gels Important Samples containing 250 mM NaCl 100 mM KCI 10 mM acetate ions or 10 mM EDTA i e certain restriction enzyme and PCR buffers will cause loss of resolution on E Gel agarose gels To obtain the best results dilute samples which contain high salt levels 2 to 20 fold 1 Take the volume listed below for the type of sample you wish to dilute Source Sample Volume Restriction Digest fragment size gt 1 kb 1 uL Restriction Digest fragment size lt 1 kb 5 10 uL PCR 1 5 uL Digest of 500 ng 1 ug DNA in 20 uL PCR reaction size of 50 uL 2 Dilute samples as described below for the type of gel you are using Gel Type Dilution E Gel CloneWell agarose Dilute samples with loading gel buffer deionized water or TE to a final volume of 20 25 uL E Gel single comb gel E Gel double comb
82. fetechnologies com egels System The High Throughput E Gel Electrophoresis System is compatible for use with components multichannel pipettors or automated liquid handling systems The system consists of the following components E Gel 96 gels see below and next page Mother E Base and Daughter E Base page 15 E Holder Platform page 16 E Editor 2 02 Software page 16 Applications E Gel 96 agarose gels are suitable for analyzing multiple samples PCR products Restriction digests RT PCR reactions Library screenings SNPs analysis 13 E Gel 96 Agarose Gels E Gel 96 gels Diagram of E Gel 96 cassette 14 E Gel 96 gels are self contained pre cast agarose gels that include agarose a proprietary buffer system ethidium bromide or SYBR Safe DNA stain and electrodes packaged inside a dry disposable UV transparent cassette Each E Gel 96 gel contains 96 sample lanes and 8 marker lanes in a patented staggered well format see figure on the next page The wells of the E Gel 96 gel are compatible with the standard 96 well plate format for automated loading See page 22 for product specifications In addition each E Gel 96 cassette is labeled with an individual barcode to facilitate identification of the gel using commercial barcode readers page 87 The lane numbers are labeled with fluorescent dye that transfers to the image and allows tracking of your samples during photo documentation of the gel
83. for Biomolecules Screening standard 96 well plate format of the E Base fits on most robotic platforms allowing the loading and electrophoresis of gels on the E Base directly on the robot E Gel iBase specifications Dimensions Weight Safety Temperature Built in Features The specifications for E Gel iBase are listed below 18 4 cm x 11 cm x 5 75 cm 500 g UL listed and CE certified Ambient 15 C to 40 C Alarm light LED LCD Display E Gel Safe Imager Real time Transilluminator Specifications Viewing surface dimensions Case dimensions Amber filter dimensions Weight of Safe Imager Weight of Filter Electrical Requirements Temperature Built in Features LED life LED Specifications Included accessories Adapter Specifications The specifications for the E Gel Safe Imager Real time Transilluminator are listed below 62 x 77 mm 200 x 110 x 43 mm 121 x 138 x 31 mm 243 g 55g 48 VDC 0 8 A max Ambient 5 C to 40 C LED light 50 000 hours Array of 12 high power LEDs emitting at 480 5 nm The LEDs used radiate less than 10 Lumens each at 200 mA Amber filter unit and viewing glasses for viewing results Use only the UL Listed adapter supplied with the starter kit or with the E Gel iBase Power System Input Output 100 240 VAC 50 60Hz 1A 48 VDC 0 8 A 23 Product Specifications Continued E Gel PowerBase v 4 specifications E Ge
84. for a diagram of the bases Each Mother E Base has a pwr prg power program button right side and a time button left side on the lower right side of the base The lower left side of each Mother E Base contains a light LED and a digital display The gel cassette is inserted into the two electrode connections The Mother E Base is connected to an electrical outlet with the electrical plug The E Base is pre programmed with 2 programs specific for each gel type as described below Program Gel Type Run Parameters EG E Gel 96 Time 12 minutes EP E PAGE 96 lime 14 minutes EG E Gel 48 1 and 2 Time 20 minutes EG E Gel 48 4 Time 17 minutes EP E PAGE 48 Time 23 minutes Mother E Base to electrical outlet electrode electrode 22 PWr prg ose button Pi digital display 15 E Base Power Supply Continued Daughter E Base E Holder Platform E Editor 2 02 software 16 The Daughter E Base is similar to the Mother E Base except the Daughter E Base does not have an electrical cord and cannot be connected to an electrical outlet The Daughter E Base is connected to a Mother E Base or to another Daughter E Base already connected to a Mother E Base Once connected to a Mother E Base each Daughter E Base is designed to function independently of the Mother E Base or other Daughter E Bases Mother E Base Daughter E Base Mother E Base
85. g Troubleshooting The table below provides solutions to some problems that you may encounter with E Gel single comb and double comb gels No current Copper contacts in the Make sure the copper contacts in the base are intact base are damaged Expired or defective gel Use fresh gel cassette Use properly stored gels before the cassette specified expiration date E Gel cassette is not Remove cassette and reinsert a steady red light illuminates on properly inserted in base the base when the cassette is correctly inserted and power is on Incorrect adaptor used Use only UL Listed Class 2 Direct Plug in Adaptor included with the E Gel iBase and PowerBase Poor resolution or Sample is overloaded Load 20 100 ng of sample DNA per band Less DNA is required smearing of bands since E Gel agarose gels are thinner High salt concentration Dilute your high salt samples as described on page 26 Very low volume of Avoid introducing bubbles while loading the samples Bubbles sample loaded or sample will cause band distortion was not loaded properly Load the recommended sample volume based on the gel type and loading method For proper band separation keep sample volumes uniform Load deionized water or TE into any empty wells Gel was not For best results run the gel within 15 minutes of sample electrophoresed loading immediately after sample If you cannot run the gel immediately after sample loading use loading the Two S
86. g pUC19 EcoR I cut 50 ng pUC19 uncut 50 ng E Gel Low Range DNA Marker 350 ng Low DNA Mass Ladder 470 ng O CON A FTF FP WN HB p p ek Ne oO 45 Troubleshooting Troubleshooting The table below provides solutions to some problems that you may encounter with E Gel with SYBR Safe agarose gels No current Copper contacts in the base Make sure the copper contacts in the base are intact are damaged due to improper use Expired or defective gel Use fresh gel cassette Use properly stored gels before the cassette specified expiration date E Gel with SYBR Safe Remove cassette and reinsert a steady red light illuminates on cassette is not inserted the base when the cassette is correctly inserted and power is properly into a base on Use only UL Listed Class 2 Direct Plug in Adaptor included with the E Gel iBase and PowerBase Very low volume of sample Avoid introducing bubbles while loading the samples Bubbles loaded or sample was not will cause band distortion loaded properly Load the recommended sample volume based on the gel type and loading method For proper band separation we recommend keeping sample volumes uniform Load deionized water or TE into any empty wells Gel was not electrophoresed For best results run the gel within 15 minutes of sample immediately after sample loading loading If you cannot run the gel immediately after sample loading use the Two Step Loading method
87. garose gel electrophoresis system is a complete bufferless system for agarose gel electrophoresis of DNA samples The major components of the system are e E Gel pre cast agarose gels e Electrophoresis bases E Gel pre cast agarose gels are self contained gels that include electrodes packaged inside a dry disposable UV transparent cassette The E Gel agarose gels run in a specially designed device that is a base and power supply combined into one device two bases are available for running E Gels the new iBase system and the original economical E Gel Powerbase Using E Gel agarose gels for electrophoresis of DNA samples offer the following advantages Provides fast safe consistent high resolution electrophoresis Eliminates the need to prepare agarose gels and buffers and to stain gels Compatible with most commercially available robotic systems for high throughput agarose gel electrophoresis Available in a variety of agarose percentages well formats and throughput capacities to suit your applications Offered with a number of different DNA gel stains to accommodate your application Includes E Gel CloneWell and E Gel SizeSelect gels to accelerate and simplify DNA gel purification and improve cloning results Three categories of E Gel agarose electrophoresis systems are available based on your throughput requirements Low Throughput E Gel Electrophoresis System designed for electrophoresis of 8 16 DNA samp
88. gel select program EG and then reinsert the gel in the base 89 Loading E Gel 48 and 96 Gels Continued Method of Loading We recommend the following methods of sample loading based on the gel type Samples Gel Type Method of Loading E Gel 48 Manual multichannel pipettor load samples into alternate wells of the gel followed by a second round of loading into the remaining wells or robotic loading devices 8 or 12 tip E Gel 96 Manual multichannel pipettor or robotic loading devices 8 12 or 96 tip Total Sample The recommended total sample volume for each gel type is listed in the table below Volume Note For best results keep all sample volumes uniform If you do not have enough samples to load all wells of the gel load an equal volume of buffer containing the same salt concentration as samples into any empty wells Gel Type Total Sample Volume E Gel 48 gel 15 uL E Gel 96 gel 20 uL One Step Loading Load DNA samples into the gel as described below see page 84 for sample preparation Method Avoid introducing bubbles while loading as they will cause the bands to distort The gel should be loaded within 15 minutes of removal from its plastic pouch Load prepared samples into each well e For E Gel 48 Gels Load 15 uL of prepared sample into sample wells Load 15 uL of sample buffer containing the same salt concentration as your sample into any remaining empty wells e For E Gel 96 Gels Load 20 uL
89. ger Blue Light Transilluminator Blue light transilluminators available from other manufacturers are also compatible for use with E Gel with SYBR Safe e Standard 300 nm UV transilluminator e Imaging systems such as laser based scanners equipped with an excitation source in the UV range or between 470 530 nm Note If you plan to excise bands for cloning use a blue light transilluminator to visualize your DNA UV light sources in combination with SYBR Safe stain could lead to reduced cloning efficiencies Using a blue light transilluminator will also minimize your personal UV exposure Photograph E Gel with SYBR Safe using a CCD camera or a laser based scanner For photographing gels refer to page 127 to determine the optimal filter sets to use or contact the instrument manufacturer for advice Do not use ethidium bromide filters that block light above 500 nm for photographing E Gel with SYBR Safe While yielding similar sensitivities to ethidium bromide SYBR Safe is somewhat dimmer yet with a lower background than ethidium bromide As a result a slightly longer exposure time or higher gain setting may be necessary Results with E Gel 48 Gels 196 E Gel 48 Gel Results obtained using a 1 E Gel 48 gel is shown in the figure below The gel was electrophoresed for 23 minutes using the standard conditions described in this manual and imaged using a KODAK EDAS290 system You can use a mini transilluminator to view th
90. ght and beeping rapid beeping for 30 seconds followed by a single beep every minute signals the end of the run The LCD displays Run Complete Press Go Press and release the Go button to stop the beeping The LED shows a steady red light and the LCD display shows the most recent program and settings Remove the E Gel EX cassette from the iBase device You are now ready to proceed to imaging or any other application with the gel To open the E Gel EX cassette for excision of DNA fragments or for blotting see page 119 for details Electrophoresis can be interrupted at any time by pressing and releasing the Go button to stop the current A flashing red light indicates that the current is stopped and the digital display flashes the message Press GO to Run Hold Go to Reset to indicate that the run was interrupted You can remove the gel from the iBase device to check the progress of the run then Continue the run from the point at which it was stopped Reinsert the gel and press and release the Go button The light changes to a steady green and the LCD display shows the count down time The run time but not the program can be adjusted before continuing the run Cancel the interrupted run Press and hold the Go button for a few seconds The LCD display resets and returns to Ready Mode A new program and run time can be selected to rerun the gel Running RNA Samples on E Gel EX Agarose Gels E Gel EX agarose gels for RN
91. hown in the figure below The gel was electrophoresed for 12 minutes using the standard conditions described in this manual and imaged using a KODAK EDAS120 system You can use a mini transilluminator to view the bands You can vary the amount of markers loaded to improve gel imaging Note The wells of the E Gel 96 gel are staggered DNA bands migrate between adjacent wells in the row below For example the bands of lane A2 will migrate between wells B1 and B2 The box highlights a lane The gel contains the following samples Lane Sample 1 279 1 kb PCR product 100 ng 4 5 6 10 11 12 3 kb PCR product 100 ng 7 8 9 9 kb PCR product 100 ng M E Gel High Range DNA Marker 101 Results with E Gel 96 Gels Continued 2 E Gel 96 Gel 102 Results obtained using a 2 E Gel 96 gel are shown in the figure below The gel was electrophoresed for 12 minutes using the standard conditions described in this manual and imaged using a KODAK EDAS120 system You can use a mini transilluminator to view the bands You can vary the amount of markers loaded to improve gel imaging MEL ELT n Wara L 2 4 The gel contains the following samples Lane Sample 1 2 3 10 11 12 125 bp PCR product 100 ng 4 5 6 240 bp PCR product 100 ng 7 8 9 1 kb PCR product 100 ng M E Gel Low Range Quantitative DNA Ladder Using E Editor 2 02 Software Introduction The E Editor 2 02 software for Windows allows
92. ht markers page 49 DNA sample capacity Refer to the following table to determine the amount of sample to run on an E Gel EX agarose gel If you are unsure how much sample to use test a range of concentrations to determine the optimal concentration for your particular sample Note Exceeding the maximum amount of DNA results in poor resolution For best results keep all sample volumes uniform If you do not have enough samples to load all the wells of the gel load an identical volume of deionized water into any empty wells Single DNA Multiple DNA Optimal Maximum Sample 7o Agarose Sample Amount Amount 1 1 100 ng 1 50 ng band 3 25 ng 250 ng 2 1 300 ng 1 100 ng band 5 150 ng 500 ng 4 1 300 ng 1 100 ng band 5 200 ng 500 ng The proprietary fluorescent nucleic acid stain in E Gel EX agarose gels is more sensitive Note than ethidium bromide For cloning purposes be sure to load enough DNA for your application as quantities of DNA that are sub optimal for cloning can still produce a strong signal Preparing samples Use a total sample volume of 20 pL for each well Prepare your samples by adding E Gel Sample Loading Buffer or deionized water to your DNA sample to bring the total volume to 20 yL For samples that are in a high salt buffer refer to page 26 48 Molecular Weight Markers DNA molecular weight markers Product E Gel EX 1 agarose E Gel EX 2 agarose E Gel EX 4 agarose We r
93. ide gels 8 gels G7008 01 E Gel 96 2 with Ethidium Bromide gels 8 gels G7008 02 Electrophoresis Bases DNA Molecular Weight Markers E Gel iBase USB Mini Cable E Holder The following electrophoresis bases are available from Life Technologies for electrophoresis of E Gel agarose gels The E Gel iBase Power System Cat nos G6400 G6400EU G6400UK is used for electrophoresis of E Gel CloneWell E Gel EX E Gel SizeSelect single comb and double comb gels The E Gel PowerBase v 4 available only in starter kits is used for electrophoresis of E Gel single comb and double comb gels The Mother E Base Cat no EB M03 is used for electrophoresis of one E Gel 48 or 96 gel The Daughter E Base Cat no EB D03 attaches to the Mother E Base and is used for electrophoresis of two or more E Gel 48 or 96 gels A large variety of DNA molecular weight markers for use with E Gel agarose gels are available from Life Technologies The recommended DNA marker for each gel type and ordering information is provided on pages 28 38 60 49 85 Cat No 10488095 E Gel 25 bp DNA Ladder E Gel 50 bp DNA Ladder Cat No 10488099 E Gel iBase USB Mini Cable Cat no G6300 is used to download firmware upgrades for the E Gel iBase Power System from www lifetechnologies com The E Holder Platform is used to hold an E Gel 48 or 96 gel in place for robotic loading Cat no EH 03 The E Holder is not a po
94. illuminator significantly reducing DNA damage that can reduce cloning efficiency Dispose of E Gel EX and E Gel SizeSelect agarose gels as hazardous waste in the same manner as ethidium bromide containing gels Contact your safety office or local municipality for appropriate disposal in your community When bound to nucleic acids the proprietary nucleic acid stain in E Gel EX and E Gel SizeSelect agarose gels has fluorescence excitation maxima at 490 nm and an emission maximum at 522 nm see figure below Normalized fluorescence excitation and emission spectra of proprietary DNA gel stain in E Gel EX and E Gel SizeSelect agarose gels determined in the presence of DNA Excitation Emission Fluorescent excitation Fluorescence emission 350 450 550 650 Wavelength nm Detect DNA bands stained with proprietary DNA gel stain using a blue light transilluminator a standard UV transilluminator or a laser based scanner For photographing gels a special filter may be required refer to page 127 for more information Using a blue light transilluminator method dramatically reduces DNA damage As a result cloning efficiency can improve ten to thousand fold For more information go to https www lifetechnologies com us en home life science dna rna purification analysis nucleic acid gel electrophoresis dna stains sybr safe html Filter Selection Guide Filter Selection Guide Use the filter rec
95. inator to view the bands Note You may vary the amount of markers loaded to improve photography of the gel 0 8 double comb Results obtained using a 0 8 double comb E Gel gel are shown below 10 pL loaded in gel M lanes 20 uL loaded in sample lanes Digestion of pUC18 and pcDNA 3 1 are as described for the 0 8 single comb gel page 32 Lane Sample High DNA Mass Ladder 200 ng pcDNA3 1 Nco I cut 150 ng pcDNA3 1 uncut 120 ng pUC18 Pst I 60 ng Low DNA Mass Ladder 125 ng 1 kb PCR fragment 3 kb PCR fragment 5 kb PCR fragment High DNA Mass Ladder 200 ng CIE er ewe meer ses Lanes 9 16 contain samples as described for Lanes 1 8 Agarose 3P DoubleComb OST OP Oe ee SS de 2 double comb Results obtained using a 2 double comb E Gel gel are shown below 10 uL loaded in M gel lanes 20 uL loaded in sample lanes Digestion of pUC18 and pcDNA 3 1 are as described for the 0 8 single comb gel page 32 Lane Sample 1 Kb Plus DNA Ladder 300 ng peDNA 3 1 Nco I cut 150 ng pcDNA 3 1 uncut 120 ng pUC18 Pst I 60 ng Low DNA Mass Ladder 125 ng 500 kb PCR fragment 1 kb PCR fragment 2 kb PCR fragment 1 Kb Plus DNA Ladder 300 ng Lanes 9 16 contain samples as described for Lanes 1 8 n rs Vari Doublet ombi zd Qu os Ip e e Ss Jd dem Note We have adjusted the brightness and contrast to improve the reproduction quality of the E Gel gel images in this manual 35 Troubleshootin
96. ining and is packaged inside an UV transparent cassette To create a patented bufferless system each E Gel single comb and double comb cassette contains two ion exchange matrices IEMs that are in contact with the gel and electrodes The IEMs supply a continuous flow of ions throughout the gel resulting in a sustained electric field required for running the gel see figure below See page 22 for product specifications Running gel Cathode D E Lower IEM Upper IEM The lower IEM near the The upper IEM near Upper IEM Anode anode contains Tris cations the cathode contains and ethidium bromide acetate anions OH AC Lower IEM Cut Trist E Gel CloneWell and E Gel SizeSelect pre cast agarose gels provide a novel way to purify DNA bands and offer the following advantages Saves time by not requiring additional gel purification steps after electrophoresis Simplifies DNA recovery since purified DNA is removed directly from the well with a pipette Improves cloning results by minimizing UV related DNA damage leading to more colony forming units than other cloning methods Supplied as precast 0 8 E Gel CloneWell or 2 E Gel SizeSelect agarose gels in the familiar E Gel format allowing fast safe consistent and high resolution separation of small and large DNA fragments For details on E Gel CloneWell agarose gels see page 59 For details on E Gel SizeSelect agarose gel
97. is System consists of the following components E Gel CloneWell E Gel EX E Gel NGS E Gel SizeSelect E Gel with SYBR Safe E Gel single comb and E Gel double comb pre cast agarose gels next page E Gel iBase Power System or E Gel PowerBase v 4 The E Gel iBase Power System and E Gel PowerBase v 4 are a base and a power supply in one device These power systems connect directly to an electrical outlet using the adaptor supplied with the base page 7 9 E Gel Safe Imager Real time Transilluminator specifically designed for use with E Gel EX E Gel NGS E Gel SizeSelect and SYBR Safe stained DNA gels run on E Gel iBase Power System not suitable for viewing ethidium bromide stained gels page 18 E Gel Opener page 10 Note The E Gel Base previously available from Invitrogen can be used for electrophoresis of E Gel with SYBR Safe E Gel single comb and double comb agarose gels page 130 E Gel agarose gels are suitable for analyzing or purifying PCR products Restriction digests RT PCR reactions Low Throughput E Gel Well Formats E Gel single comb and double comb gels Features of E Gel CloneWell and SizeSelect agarose gels The E Gel single comb and double comb gels are bufferless gels containing electrodes embedded in the agarose matrix Each gel contains an ion generating system TAE buffer system a pH balancing system and ethidium bromide for DNA sta
98. isplay will show the default time e g 12 minutes or the last programmed time 3 To begin electrophoresis press and release the power button located on the lower right corner of the mother base see figure below and daughter base The red light will change to a green light while the run is in progress E Gel 96 mother base to electrical outlet electrode connection E Gef 96 gel barcode electrode I connection oy power vd button timer e button E Gel 96 3 mother base Y light LED 4 digital timer display While the run is in progress you can add to the run time by pressing the time button To interrupt or stop a run in progress see next page 4 The mother base will signal the end of the run with a flashing red light and rapid beeping for 2 minutes followed by a single beep every minute The digital display will show the elapsed time up to 19 minutes with a negative sign since the end of the run 5 Press and release the power button to stop the beeping The light will turn to a steady red and the digital display will show the last time setting 6 Remove the gel cassette from the mother base and daughter base You are now ready to capture an image of the gel Note The bands in the gel will diffuse within 20 minutes 106 Using E Gel 96 Mother Daughter Base Continued Interrupting an You can interrupt an electrophoresis run at any time by pressing and releasing the power El
99. itation we recommend using the Qubit fluorometer Cat no Q32857 with the Quant iT dsDNA HS Assay Kit Cat nos Q32851 or Q32854 An accurate working range can accommodate up to 40 ng of DNA in the final Qubit reaction mixture For spectrophotometric quantitation of recovered DNA we recommend buffer exchange using the PureLink PCR Micro kit Cat nos K310010 or K310050 81 Troubleshooting No current Copper contacts in the base Make sure the copper contacts in the base are intact are damaged due to improper use Expired or defective gel Use fresh gel cassette Use properly stored gels before the cassette specified expiration date E Gel SizeSelect cassette is Remove cassette and reinsert a steady red light illuminates not inserted properly intoa on the base when the cassette is correctly inserted and power base is on Incorrect adaptor used Use only UL Listed Class 2 Direct Plug in Adaptor included with the E Gel iBase Poor resolution Sample is overloaded Do not load more than the recommended amount of DNA smeared bands sample per band see page 48 i High salt concentration Dilute your high salt samples as described on page 26 Aberrant pre run step Do not pre run E Gel SizeSelect agarose gels Very low volume of sample Avoid introducing bubbles while loading the samples loaded or sample was not Bubbles will cause band distortion loaded properly Load the recommended sample volume based on the gel type
100. ith the iBase Power System Installing the If using only the iBase Power System attach the power cord of the iBase to the power iBase M Power inlet and then to the electrical outlet Use only properly grounded AC outlets and cords System Installing the If using the iBase Power System and Safe Imager Real time Transilluminator iBase Power 1 Place the iBase directly onto the E Gel Safe Imager Real time Transilluminator so System with the that the legs of the iBase fit directly into the grooves of the Safe Imager as shown in Safe Imager the image below 2 Plug the short electrical cord of the E Gel Safe Imager Real time Transilluminator a into the power inlet of the iBase b 3 Plug the connecting end of the power cord with the transformer into the back inlet of the Safe Imager c and connect the power cord to the electrical socket To power outlet 61 Using E Gel CloneWell Agarose Gels with the iBase Power System Continued Insertacassettein 1 Plug the iBase device into an electrical outlet using the adaptor plug on the base the iBase Power 2 Open the package and remove the gel Do not remove the combs until you start System loading the samples 3 Slide the cassette into the two electrode connections on the iBase device Press down on the left side of the cassette to secure it into the iBase device The two electrodes on the right side of the gel cassette must be in contact with the two ele
101. kb 800 bp 10 kb 100 bp 5 kb 100 bp 2 kb 20 bp 500 bp 100 bp 5 kb 100 bp 2 kb 100 bp 6 kb 50 bp 2 kb 1 kb 10 kb 100 bp 2 kb 400 bp 10 kb 90 bp 3 kb 10 bp 400 bp 1 kb 10 kb 100 bp 2 kb Gel Selection Continued Apparatus The table below lists the power systems compatible with the various types of E Gel compatibility agarose gels Gel Type E Gel iBase Power E Gel PowerBase Mother and Daughter E Base System v 4 Integrated Power Supply E Gel CloneWell Y E Gel with SYBR Safe Y Y N E Gel EX Y N N E Gel NGS Y N N E Gel SizeSelect Y N N E Gel single comb and double comb with Y Y N ethidium bromide E Gel 48 26 Gel N N Y The E Gel iBase Power System is compatible with the E Gel Safe Imager Real time Transilluminator The E Gel PowerBase v 4 is compatible with the Safe Imager 2 0 Blue Light Transilluminator Gel Selection Continued Advantages of E Gel EX agarose gels Advantages of E Gel NGS agarose gels Advantages of E Gel with SYBR Safe agarose gels Advantages of E Gel CloneWell agarose gels Advantages of E Gel SizeSelect agarose gels E Gel EX pre cast agarose gels have the following features General use agarose gels containing a proprietary fluorescent nucleic acid stain High sensitivity with detection down to 1 ng band of DNA Compatibility with blue light transillumination to dramatically reduce DNA damage and maximize cloning efficiency
102. l agarose gels with the E Gel Base see page 132 Install the iBase If using only the E Gel iBase Power System attach the power cord of the iBase device to the power inlet and then to an electrical outlet Use only properly grounded AC outlets Power System and cords alone Install the iBase If using the E Gel iBase Power System in conjunction with the Safe Imager Real time Power System and Transilluminator Safe Imager 1 Place the iBase device directly onto the Safe Imager transilluminator so that the legs of the iBase device fit directly into the grooves of the Safe Imager transilluminator 2 Plug the short electrical cord of the Safe Imager transilluminator a into the power inlet of the iBase device b 3 Plug the connecting end of the power cord with the transformer into the back inlet of the Safe Imager transilluminator c and connect the power cord to the electrical socket Transilluminator To power outlet 39 Using E Gel NGS and E Gel with SYBR Safe Gels with the iBase Power System Continued Insert a cassette in the iBase Power System Load the E Gel agarose gel 40 1 Open the package and remove the gel Do not remove the comb until you start loading the samples Slide the cassette into the two electrode connections on the iBase device Press on the left side of the cassette to secure it in the iBase device The two electrodes on the right side of the gel
103. l cassette in the E Holder Align the bottom left end of the cassette in the lower left alignment corner of the E Holder as shown in the figure below E Halder E33 E3 D EE EE E EE LE riri E Aa CU CE cm cac c Beer ems Eee Be Bea E Gel 26 gel Alignment comer Set up your robotic system to load samples into the E Gel 48 or 96 gel placed on an E Holder Program your robotic system to load the samples approximately 5 minutes before the previous electrophoresis run is complete This will ensure that the loaded gel from the E Holder will be placed onto an E Base within the recommended time of 15 minutes 95 Visualizing E Gel 96 with SYBR Safe Agarose Gels Introduction Viewing E Gel with SYBR Safe Imaging E Gel with SYBR Safe Required Filters Important Exposure Time and Gain Setting 96 Bound to nucleic acids SYBR Safe DNA gel stain has fluorescence excitation maxima at 280 and 502 nm and an emission maximum at 530 nm Use a blue light or UV light transilluminator to view the gel a filter is required to photograph the gel your standard ethidium bromide filter may not be appropriate see below View E Gel with SYBR Safe using these instruments e Blue light transilluminator The Safe Imager 2 0 Blue Light Transilluminator Cat no G6600 is designed specifically for use with SYBR Safe stained DNA gels Refer page 17 for instructions on using the Safe Ima
104. l iBase Adaptor specifications E Gel PowerBase Adaptor specifications E Gel Base specifications 24 The specifications for E Gel PowerBase V 4 are listed below Dimensions 12 5 cm x 13 cm x 13 5 cm Weight 1 19 Ibs 540 g with adaptor Safety UL listed and CE certified Temperature Ambient 15 C to 40 C Built in Features Alarm light LED The E Gel iBase is designed for use with an adaptor included with the iBase Use only the UL Listed original adapter supplied Input 100 240 VAC 50 60Hz 1A Output 48 VDC 0 8 A The E Gel PowerBase v 4 is designed for use with an adaptor included with the PowerBase Use only UL Listed Class 2 Direct Plug in Adaptor included with the PowerBase Input and Output supplied by the adaptor are shown in the table below US and Canada 110 120 V AC 60 Hz 12V DC 880 mA Europe 220 240 V AC 50 Hz 12 V DC 880 mA The specifications for E Gel Base are listed below Dimensions 12 5 x 13 x 3 5 cm Weight 3 18 oz 90 g Temperature Ambient 15 C to 40 C Methods General Guidelines Introduction Materials needed General guidelines Important Loading E Gel agarose gels Loading buffer For optimal results follow these general guidelines for preparing your DNA sample For specific details related to running each type of E Gel agarose gel refer to the section for that particular gel type DNA sample Molecular weight markers Optional E
105. les per gel Medium Throughput E Gel Electrophoresis System designed for electrophoresis of 48 DNA samples per gel This system is compatible for use with multichannel pipettors or automated liquid handling systems High Throughput E Gel Electrophoresis System is designed for electrophoresis of 96 DNA samples per gel This system is compatible for use with multichannel pipettors or automated liquid handling systems Gel Selection Choosing a gel for To obtain the best results for your application it is important to choose the correct your application agarose percentage and well format The table below lists the various types of gel and resolution for each gel type Gel Type E Gel EX E Gel NGS E Gel single comb with ethidium bromide E Gel with SYBR Safe E Gel CloneWell E Gel SizeSelect E Gel double comb with ethidium bromide E Gel 48 Gel E Gel Gel 10 sample 1 marker 10 sample 1 marker 8 sample 1 marker 8 sample 1 marker 16 sample 2 marker 48 sample 4 marker 96 sample 8 marker 20 uL 20 uL 20 uL 20 uL 20 25 ul 20 25 uL 20 uL 10 uL 15 uL 15 uL 20 uL 20 uL Wells compatible for loading with a multichannel pipettor 2 0 8 0 8 1 2 2 4 1 2 2 0 8 2 0 8 2 1 2 4 1 2 No Sample Sample Run Length Agarose Resolution Sind Loading Wells Volume 190 100 bp 5 kb 50 bp 2 kb 800 bp 10
106. lts in poor resolution For best results keep all sample volumes uniform If you do not have enough samples to load all the wells of the gel load an identical volume of deionized water into any empty wells Single DNA Multiple DNA Optimal Sample Band Bands Amount 20 400 ng 500 700 ng 50 200 ng Prepare your samples as described below 1 Prepare the samples by adding deionized water to the required amount of DNA to bring the total sample volume to 20 25 pL Prepare the DNA molecular weight marker by adding deionized water to the required amount of DNA to bring the total sample volume to 5 10 pL Instead of water you may use a loading buffer to prepare samples or DNA molecular weight marker See page 25 for more details Do not use a tracking dye to avoid masking the bands For samples that are in a high salt buffer refer to page 26 59 Molecular Weight Markers DNA molecular weight markers 60 We recommend using the following DNA molecular weight markers for E Gel CloneWell 0 8 with SYBR Safe to obtain good resolution Note Supercoiled DNA molecular weight markers may produce a slightly fuzzy pattern when run on E Gel CloneWell 0 8 with SYBR Safe agarose gels Markers Amount Used E Gel 1 Kb Plus DNA Ladder 10488 090 Load 500 700 ng E Gel High Range DNA Marker 12352 019 rire a volume 1 Kb Plus DNA Ladder 10787 018 High DNA Mass Ladder 10496 016 Using E Gel CloneWell Agarose Gels w
107. luorescent Green 260 2034 Express Investigator Plus Luminary FOTODYNE FOTO Analyst Minivisionary FOTODYNE UV Fluorescent Green 2162 4289 FOTO Analyst Apprentice FOTODYNE UV Fluorescent Green 12162 2535 FOTO Analyst Luminary FOTODYNE UV Fluorescent Green 1560 2056 FCR 10 Polaroid UV 113 4218 FUJI FLA 3000 FUJI Film 473 nm 520LP BioDocIt AC1 EC3 BioSpectrum UVP 302 nm SYBR Green 38 0219 01 or SYBR Gold 38 0221 01 Gel Logic Kodak UV 535 nm WB50 Syngene Instruments Syngene UV 500 600 nm Shortpass filter 127 Appendix F E Gel iBase Power System Downloading Firmware Upgrades for the E Gel iBase Introduction iBase Updater Firmware Update Troubleshooting 128 Instructions are provided below to upgrade the firmware on the E Gel iBase Power System Firmware upgrade requires installation of the iBase Updater program 1 Download the iBase Updater file iBaseUpdater zip from Life Technologies at www invitrogen com ibase Extract the iBaseUpdater exe file from the zip folder Double click the iBaseUpdater exe file and follow the instructions to install the program To launch the iBase Updater click on Start gt All Programs gt Invitrogen gt Updaters and select iBase Updater pe e e x Disconnect the electrical plug of the iBase device from the electrical outlet Make sure the USB cable is not connected Press and hold the Go button red button
108. may be impaired if the equipment is used in a manner not specified by Life Technologies The device must be connected to a mains socket outlet with protective earthing connections Ventilation requirements no special requirements The E Gel iBase Power System complies with part 15 of the FCC rules Operation of the device is subject to the following two conditions e The device may not cause harmful interference e The device must accept any interference received including interference that may cause undesired operation Life Technologies Israel Ltd a Life Technologies company is the manufacturer and owner of the UL file For more information contact Life Technologies Israel Ltd 12 Hamada St Rehovot Israel 76703 For more information contact Technical Support see page 110 The E Gel PowerBase complies with the Underwriters Laboratories Inc regulation and is listed under file no E189045 in the US and Canada This device complies with part 15 of the FCC Rules Operation is subject to the following two conditions e This device may not cause harmful interference e This device must accept any interference received including interference that may cause undesired operation 115 Explanation of Symbols and Warnings Continued E Gel iBase CE LISTED E189045 FC EN60825 1 A Caution 116 The E Gel iBase Power System and E Gel Safe Imager Real time Transilluminator comply with the U
109. mples E Gel 48 gels lanes Sample leaking from Sample is overloaded Be sure to load the recommended volume of sample per the wells well Use Use the Two Step Loading method page 147 Two Step Use the Two Step Loading method page 147 method page 117 Wells damaged during comb Be sure to remove the comb gently without damaging the removal wells Over run the gel or Accidentally selected a Select EG if you are using E Gel 96 gels For E Gel 48 gels need more time to different program select EG and then manually change the time to 20 minutes run gel If the wrong program is selected by accident and you are well into the run check the gel to see where the loading dye is running Estimate the amount of time remaining and then manually stop the run Failure Mode Defective cassette Disconnect the base and replace gel cassette with a fresh gel indicated by a steady cassette red and continuous Press and release the power button to return to Ready rapid beeping and Mode flashing ER on an m EN Cold cassette Use a room temperature cassette stored at room temperature Avoid storing gel cassettes at 4 C Improper operating Use the E Base at room temperature 20 25 C conditions Speckles visible Dust fluorescing in same Make sure gel is clean before imaging SYBR Safe gel wavelength as SYBR Safe High background No filters or wrong filter set Refer to page 127 to determine the optimal filter
110. mples of the same E Gel 2 with SYBR Safe recorded with different imaging methods are shown An example of DNA samples run on an E Gel 1 2 with SYBR Safe is shown below Samples were loaded in a total volume of 20 uL and visualized on a standard 312 nm UV transilluminator Photographs were taken using the MiniBis photo documentation system from DNR and the SYBR Safe photographic filter using an exposure time of 1 8 sec m o b Sample High DNA Mass Ladder 130 ng E Gel High Range DNA Marker 200 ng 1 kb PCR product 100 ng 3 kb PCR product 200 ng 9 kb PCR product 200 ng 1 Kb plus DNA Ladder 500 ng 1 Kb plus DNA Ladder 500 ng pBR322 EcoR I cut 100 ng pBR322 uncut 100 ng E Gel with SYBR Safe d pUC19 EcoR I cut 50 ng invitrocen 11 E Gel High Range DNA Marker 200 ng 12 High DNA Mass Ladder 130 ng 1 2 Agarose GP 12345 a ee a Ct EEEN E CCH I WOON A TF WN e An example of DNA samples run on an E Gel 2 with SYBR Safe is shown below Samples were loaded in a total volume of 20 uL and visualized on a standard 312 nm UV transilluminator Photographs were taken using the MiniBis photo documentation system from DNR and the SYBR Safe photographic filter using an exposure time of 1 8 sec m o b Sample Low DNA Mass Ladder 470 ng E Gel Low Range DNA Marker 350 ng 240 bp PCR product 500 ng 317 bp PCR product 700 ng 1 kb PCR product 100 ng 10
111. n time and displays a flashing red light and beeps rapidly If your band did not reach the reference line run the gel for a few more minutes until the band reaches the line Place the iBase and CloneWell on the Safe Imager to allow for monitoring during the run estimated run time Reference Line eS wee Bands reaching the Reference Line Run the gel until the band of interest reaches the Reference Line Reference Line i Lal Collecting DNA Using E Gel CloneWell Agarose Gels continued Electrophoresis of 1 bands from reference line to collection well Retrieving DNA Once the band reaches the reference line refill the second row again with sterile water until the well is full some pre filled water is lost during the run Note If more concentrated DNA is desired do not completely fill the bottom well This will result in the retrieved DNA being more concentrated Press the Go button to run the gel for the time listed in the table below until the band enters the collection well During this period of time monitor the run over a Safe Imager At the end of this run you may see the band of your interest migrating into the well Note We recommend monitoring the run in a darkened room for optimal results Small DNA amounts and low molecular weight bands may be difficult to view inside the well 1 Band Size From Reference Line to Collection Well 200 bp 1 2 minutes 400 bp 1 2 minutes 800 b
112. nderwriters Laboratories Inc regulation and the European Community Safety requirements Operation of the E Gel iBase Power System and E Gel Safe Imager Real time Transilluminator are subject to the following conditions Indoor use Altitude below 2 000 meters Temperature range 5 to 40 C Maximum relative humidity 80 Installation categories over voltage categories II Pollution degree 2 Mains plug is a disconnect device and must be easily accessible Do not attempt to open the iBase or Safe Imager device To honor the warranty iBase and Safe Imager device can only be opened and serviced by Life Technologies The protection provided by the equipment may be impaired if the equipment is used in a manner not specified by Life Technologies The device must be connected to a mains socket outlet with protective earthing connections Ventilation requirements no special requirements The E Gel iBase Power System and E Gel Safe Imager Real time Transilluminator comply with part 15 of the FCC rules Operation of the devices are subject to the following conditions e The device may not cause harmful interference e The device must accept any interference received including interference that may cause undesired operation Life Technologies Israel Ltd a Life Technologies company is the manufacturer and owner of the UL file For more information contact Life Technologies Israel Ltd 12 Hamada St Rehovot Israel 7
113. neWell Reverse run 6 E Gel 0 8 1 2 2 7 E Gel EX 1 2 8 E Gel EX 4 9 E Gel SizeSelect 2 SizeSelect 2 This mode is not compatible with E Gel 4 E Gel EX E Gel CloneWell or E Gel SizeSelect gels PRE RUN E Gel 0 8 2 0 E Gel 4 E Gel DC CloneWell 0 8 REVERSE E Gel SPEED E Gel E Gel EX E Gel EX Maximal Run Time min 40 40 20 60 20 20 20 129 Appendix G E Gel PowerBase Version 4 Using the E Gel PowerBase Version 4 Introduction Instructions are provided below to perform electrophoresis of E Gel with SYBR Safe E Gel single comb gels and double comb gels with the E Gel PowerBase v 4 Pre running using 1 Plug the PowerBase v 4 into an electrical outlet using the adaptor plug PowerBase v 4 2 Openthe package containing the gel and insert the gel with the comb in place into the apparatus right edge first Press firmly at the top and bottom to seat the gel in the base You should hear a snap when it is in place The Invitrogen logo should be located at the bottom of the base close to the positive pole See the diagram below A steady red light indicates the E Gel gel is correctly inserted Ready Mode electrical outlet light adaptor Top Bottom 3 Press and hold either button until the red light turns to a flashing green light This indicates that the 2 minute pre run has started 4 Atthe end of the pre run the current autom
114. ng the MiniBis photo documentation system from DNR and the SYBR Safe photographic filter using an exposure time of 1 8 sec 1 ide i b Y e Sample RNA Ladder native RNA Ladder native heat RNA Ladder native heat Total RNA native Total RNA native Total RNA 65 C 5 minutes Total RNA 65 C 5 minutes Total RNA formamide 65 C 5 minutes Total RNA formamide 65 C 5 minutes RNA Ladder native heat RNA Ladder native An example of mouse brain RNA samples and the 0 1 2 Kb RNA Ladder see page 110 run on an E Gel EX 2 agarose gel is shown below Samples were loaded in a total volume of 20 uL and visualized on the E Gel Safe Imager Real time Transilluminator Photographs were taken using the MiniBis photo documentation system from DNR and the SYBR Safe photographic filter using an exposure time of 1 8 sec I EPNM ZEE ZEE EI E beh HH ee eh ea ee ee r fo b T Sample RNA Ladder native RNA Ladder native heat RNA Ladder native heat Total RNA native Total RNA native Total RNA 65 C 5 minutes Total RNA 65 C 5 minutes Total RNA formamide 65 C 5 minutes Total RNA formamide 65 C 5 minutes RNA Ladder native heat Ladder formamide 65 C 5 minutes Troubleshooting No current Copper contacts in the base Make sure the copper contacts in the base are intact are damaged due to improper use Expired or defective gel
115. nning RNA Samples on E Gel EX Agarose Gels sse eene tenentes 53 Visualizing E Gel EX Agarose Cels acera edita eons datos Etude teu tid tud be da debita ices pee etn 54 Resultedisine EGel EX Ac arose Gelesen dba no niv pde Duden e b Dade ied Ee ditus 55 Results using E Gel EX Agarose Gels for RNA Samples nennen 56 TPOUBICSINO OLLIE e M 57 DNA Purification Using E Gel CloneWell Agarose Gels eccceseeeeeeeessneeeeeeeseeeeeeesseneeeeenseneeeeenss 59 Sample DrepaballODtss seeded tite epa DE A omeisarancseaiaaienmoeeMammeneniees 59 Molecular Were EM APESES us oreet remus AA debo TAE d e uri eua Gita ue TE AEA ONER 60 Using E Gel CloneWell Agarose Gels with the iBase Power System eerte 61 Collecting DNA Using E Gel CloneWell Agarose Gels eese entente enne 63 Visualizing E Gel CloneWell Agarose Gels essent rete ne tenete nennen 67 Results usine E Gel CloneWell Agarose Gels nee tier teretes tlitosetotetri s e tendi ise ne ede teure ed ar a edes 68 TOM ISS DOO IP eadeni nda tamva bitu dtc de en RV Dd e ad v creed Lud DE 69 DNA Purification Using E Gel SizeSelect Agarose Gels ceeeeeeeeeeeeeeeeeeeennnn enne 71 Sample l reparatlofisusscueetteiv du E tcx qud ba ssmeuqud tM du Mant ce pui e i dada im ce ALES 71 Molecular Were hit Markers sucesos uet ote ien assu cam ee eae usn beast aetna itas uota 22 Using Si
116. olecular weight markers and add water to any empty wells Important Do not pre run E Gel EX agarose gels Load the E Gel Load E Gel EX agarose gels within 15 minutes of opening the pouch and run within agarose gel 1 minute of loading samples Avoid introducing bubbles while loading as bubbles will cause bands to distort 1 Load 20 uL of sample per sample well see page 48 for details 2 Load 20 uL 100 250 ng of the appropriate molecular weight markers page 49 3 Load 20 uL of deionized water into any remaining empty wells 51 Using E Gel EX Agarose Gels with the iBase Power System Continued Electrophoresis Using the iBase Power System Interrupt a run on the iBase Power System 52 1 Toggle between program minutes and seconds on the iBase device by pressing the Mode button until the program blinks Use the Up Down A V buttons to select the appropriate program for your gel Gel Type Default Run Time E Gel EX 1 10 minutes E Gel EX 2 10 minutes E Gel EX 4 15 minutes Optional Change the default run time by pressing the Mode button until the minutes or seconds blink then change the values using the Up Down buttons up to the maximum run time of 20 minutes Press Go to start electrophoresis A green light indicates that the run is in progress The LCD counts down time while the run is in progress The device stops automatically when the programmed time has elapsed A flashing red li
117. ommended with your instrument below to photograph E Gel with SYBR Safe E Gel EX or E Gel SizeSelect agarose gels We have shown the most popular instruments other instruments with an excitation source in the UV range or between 470 530 nm may also be used with the proper filter Contact your instrument manufacturer for advice Instrument Manufacturer Excitation Source Emission Filter Alphalmager Alpha Innotech 302 nm SYB 500 Alphalmager HP Alpha Innotech 302 nm SYB 500 AlphaDigiDoc RT Alpha Innotech UV transilluminator Shroud Camera Stand Alpha Innotech UV transilluminator SYB 100 DE500 or DE400 light cabinet 2 17 diameter UV transilluminator SYB 500 Alpha Innotech DE500 or DE400 light cabinet 2 diameter Alpha UV transilluminator SYB 400 Innotech VersaDoc Imaging Systems Bio Rad Broadband UV 520LP Molecular Imager FX Systems Bio Rad 488 nm 530 nm BP Gel Doc Systems Bio Rad 302 nm 520DF30 170 8074 Typhoon 9400 9410 GE Healthcare 488 nm 520 BP 40 Typhoon 9200 9210 8600 8610 GE Healthcare 488 nm 526 SP Fluorlmager GE Healthcare 488 nm 530 DF 30 Storm GE Healthcare Blue fluorescence mode VDS CL GE Healthcare Transmission UV Low Ultracam Gel Imager Ultra Lum UV Yellow Filter 990 0804 07 Omega Systems Ultra Lum UV 520 nm Polaroid Camera Polaroid UV SYBR Safe Photographic Filter 527100 FOTO Analyst UV F
118. on for 2 seconds to return to Ready Mode Use a cassette stored at room temperature Avoid storing gel cassettes at 4 C Use E Gel iBase E Gel Base and E Gel PowerBase at room temperature 20 25 C Make sure gel is clean before imaging Refer to page 127 to determine the optimal filter sets to use or contact the instrument manufacturer for advice Optimize settings of your system for E Gel with SYBR Safe empirically You may need to increase the exposure time or gain setting Use IR blocking filter or emission filter with IR coating Use a blue light transilluminator such as the Safe Imager 2 0 Blue Light Transilluminator or E Gel Safe Imager Real time Transilluminator see page 112 47 Electrophoresis Using E Gel EX Agarose Gels Sample Preparation About E Gel EX E Gel EX agarose gels are pre cast 1 2 and 4 agarose gels for use with the E Gel iBase Power System E Gel EX gels have 11 wells and a novel openable format The gels contain a proprietary fluorescent nucleic acid stain excitation 490 nm emission 520 nm that can be viewed with blue light to minimize DNA damage and allows detection down to 1 ng band of DNA For optimal results using E Gel EX agarose gels follow the guidelines for preparing your DNA sample as described in this section agarose gels Materials needed DNA sample E Gel Sample Loading Buffer page 112 or deionized water for diluting samples Molecular weig
119. on systems may already include an appropriate filter for imaging the gel see page 43 for filter guidelines and contact the manufacturer for filter specifications You may use this filter in place of the amber filter unit e The Safe Imager transilluminators have a very slim design compared to UV transilluminators the distance between the camera and the gel may have to be adjusted e After viewing or documenting the results switch the Safe Imager transilluminator off Clean the Safe Imager transilluminators with a dry cloth or with water and mild soap Ethanol may also be used Avoid damaging or scratching the glass surface of the Safe Imager transilluminator with abrasive cleaners sharp instruments or harsh solvents Before cleaning the instrument disconnect it from the electrical outlet 21 Product Specifications E Gel single comb The E Gel cassette is 8 cm x 10 cm and 0 6 cm thick The thickness of the E Gel gel is and double comb gel specifications E Gel 48 Gel specifications E Gel 96 Gel specifications 22 3 mm and the volume of the gel is 20 mL Single comb gel Each well is 4 1 mm wide and the space between wells is 1 mm The running distance is 5 8 cm Each gel contains 12 lanes Double comb gel The sample well is 4 6 mm wide and the marker well is 2 5 mm wide The running distance from each comb is 2 9 cm Each gel contains two rows of 8 sample wells and 2 marker wells M The wells of the dou
120. or the Safe Imager 1 Place the iBase device directly onto the Safe Imager transilluminator so that the legs Transilluminator of the iBase device fit directly into the grooves of the Safe Imager transilluminator 2 Plug the short electrical cord of the Safe Imager transilluminator a into the power inlet of the iBase device b 3 Plug the connecting end of the power cord with the transformer into the back inlet of the Safe Imager transilluminator c and connect the power cord to the electrical socket To power outlet 73 Using SizeSelect Agarose Gels with the iBase Power System Continued Insert a cassettein 1 Openthe package and remove the gel Gently remove the combs from the upper and the iBase Power lower wells of the E Gel SizeSelect agarose gel using both hands to lift the comb System gently by rolling the comb slowly towards you Be careful to pull the comb straight up from both sides Do not bend the comb Remove any excess fluid using a pipette 2 Slide the cassette into the two electrode connections on the iBase device Press down on the left side of the cassette to secure it into the iBase device The two electrodes on the right side of the gel cassette must be in contact with the two electrode connections on the base The LED produces a steady red light to indicate that the cassette is correctly inserted Slide cassette into electrodes Press left side to secure 3 Load y
121. or last program used EG or EP 3 Select the appropriate program based on the gel Program Gel Type Run Parameters EG E Gel 96 Time 12 minutes EP E Gel 96 Time 6 minutes EG E Gel 48 1 and 2 Time 20 minutes EG E Gel 48 4 Time 17 minutes The default time is 12 minutes Change the time manually by pressing and holding the time button until the run time appropriate for your type of gel is displayed Gel Type Recommended Run Time Maximum Run Time E Gel 48 20 minutes 30 minutes E Gel 96 12 minutes or EG 20 minutes 88 Loading E Gel 48 and 96 Gels Continued Setting the Time Inserting Gel in the E Base The initial default time setting on an E Base for program EG is 12 minutes Follow instructions below to increase or decrease the time setting if desired Do not run an E Gel 96 gel for more than 20 minutes or E Gel 48 gel for more than 30 minutes To increase or decrease the default run time when no cassette is inserted on the base use the following steps 1 Connect the Mother E Base to an electrical outlet If you are using a Daughter E Base connect the Daughter E Base to the Mother E Base and then connect the Mother E Base to an electrical outlet 2 Press and release the time button located on the lower right corner of the base to view the time setting 3 Press and hold the time button to increase the time continuously When you reach the desired default time release the
122. or samples that are in a high salt buffer refer to page 26 Loading buffer is optional See page 25 for more details 27 Molecular Weight Markers DNA molecular We recommend using the following DNA molecular weight markers for different types weight markers of E Gel agarose gels to obtain good resolution Note Supercoiled DNA molecular weight markers may produce a slightly fuzzy pattern when run on E Gel agarose gels containing ethidium bromide Single comb E Gel gels E Gel 1 Kb Plus DNA Ladder 10488 090 Load 100 250 ng E Gel High Range DNA Marker 12352 019 N EUCH Kb Plus DNA Ladder 10787 018 997 500 bp DNA Ladder 10594 018 High DNA Mass Ladder 10496 016 TrackIt 1 Kb Plus DNA Ladder 10488 085 E Gel 1 Kb Plus DNA Ladder 10488 090 E Gel High Range DNA Marker 12352 019 100 bp DNA Ladder 15628 019 1 2 1 Kb Plus DNA Ladder 10787 018 High DNA Mass Ladder 10496 016 TrackIt 100 bp DNA Ladder 10488 058 TrackIt 1 Kb Plus DNA Ladder 10488 085 E Gel 1 Kb Plus DNA Ladder 10488 090 E Gel Low Range Quantitative DNA Marker 12373 031 25 bp DNA Ladder 10597 011 255 50 bp DNA Ladder 10416 014 100 bp DNA Ladder 15628 019 Low DNA Mass Ladder 10068 013 TracklIt 25 bp DNA Ladder 10488 022 TrackIt 50 bp DNA Ladder 10488 043 TrackIt 10 bp DNA Ladder 10488 019 Tracklt 25 bp DNA Ladder 10488 022 TrackIt 50 bp DNA Ladder 10488 043 4 TrackIt 1 Kb Plus DNA Ladder 10488 085 10 bp DNA Ladder 10821 014 25 bp DNA L
123. or the run over a Safe Imager transilluminator When you see the band of your interest migrating into the well press the Go button to stop the run and collect the DNA from the well using a pipette Visualizing E Gel CloneWell Agarose Gels Viewing E Gel CloneWell agarose gels Imaging E Gel CloneWell agarose gels Important Exposure time and gain setting E Gel CloneWell gels contain SYBR Safe DNA gel stain and do not have to be stained after electrophoresis SYBR Safe DNA gel stain has a fluorescence excitation maxima at 280 and 502 nm and an emission maximum at 530 nm when bound to nucleic acids Use a blue light or UV light transilluminator to view the gel a filter is required to photograph the gel your standard ethidium bromide filter may not be appropriate View E Gel CloneWell agarose gels using a Blue light transilluminator The E Gel Safe Imager Real time Trans illuminator and Safe Imager 2 0 Blue Light Transilluminator see page 112 are designed specifically for use with SYBR Safe stained DNA gels Refer to page 17 for instructions on using the E Gel Safe Imager Real time Transilluminator or Safe Imager 2 0 Blue Light Transilluminator Blue light transilluminators available from other manufacturers are also compatible for use with E Gel CloneWell gels Note UV light sources can lead to reduced cloning efficiencies e Photograph E Gel CloneWell agarose gels using a CCD camer
124. our samples Be sure to load molecular weight markers and add water to any empty wells Important Do not pre run E Gel SizeSelect agarose gels 74 Using SizeSelect Agarose Gels with the iBase Power System Continued Loading E Gel Load E Gel SizeSelect agarose gels within 15 minutes of opening the pouch and run SizeSelect within 1 minute of loading samples agarose gels Avoid introducing bubbles while loading as bubbles will cause bands to distort 1 Load 20 25 uL of sample into each well of the upper row see page 71 for details 2 Load 5 10 uL 100 250 ng of the appropriately diluted molecular weight markers page 72 into the small middle well lane M 3 Load 25 pL of deionized water into any remaining empty wells of the upper row 4 Load 25 uL of deionized water into all of the wells in the lower row collection wells Load 5 10 uL of deionized water into lane M of the lower row Load samples Load water in in wells 1 8 P any empty wells Load DNA ladder into lane M Load water in all wells p EF Gel SizeSelect invitrogen 75 Run Time Estimation for SizeSelect Agarose Gels Introduction Refer to the Run Time Table below to estimate the run time for your DNA fragment to reach the reference line and then from the reference line to reach the collection well Be sure to monitor your gel during the run If the amount of DNA is low the band may not be visible Viewing the gel in a darkened
125. ower System Safe Imager Blue Light Transilluminator and E Gel 1 Kb Plus DNA Ladder 10 gels 20 gels 10 gels E Gel iBase Power System Safe Imager Blue Light Transilluminator and E Gel 1 Kb Plus DNA Ladder 10 gels 20 gels 10 gels 10 gels 10 gels E Gel iBase Power System Safe Imager Blue Light Transilluminator and Gel Knife 10 gels 10 gels E Gel iBase Power System Safe Imager Blue Light Transilluminator and 50 bp DNA Ladder 6 gels and E Gel PowerBase 18 gels 6 gels and E Gel PowerBase 18 gels 6 gels and E Gel PowerBase 18 gels 18 gels 18 gels 18 gels The following E Gel agarose gels are available from Life Technologies Ordering information is described below Catalog no G6618 08 G6500ST G6500STEU G6500STUK G6206 01 G5218 01 G6206 02 G5218 02 G6511S5T G6511STUK G6511STEU G4010 01 G4020 01 G6512ST G6512STUK G6512STEU G4010 02 G4020 02 G401004 A25798 A25798ST A25798EU A25798UK G661002 G66125T G6612STEU G66125TUK G6000 08 G5018 08 G6000 01 G5018 01 G6000 02 G5018 02 G5018 04 G6018 08 G6018 02 Accessory Products Continued Product Quantity Catalog no E Gel 48 Gels E Gel 48 1 with Ethidium Bromide gels 8 gels G8008 01 E Gel 48 2 with Ethidium Bromide gels 8 gels G8008 02 E Gel 48 4 with Ethidium Bromide gels 8 gels G8008 04 E Gel 96 Gels E Gel 96 2 with SYBR Safe gels 8 gels G7208 02 E Gel 96 1 with Ethidium Brom
126. p 1 2 minutes 1000 bp 1 2 minutes 2000 bp 1 5 2 5 minutes 3000 bp 1 5 2 5 minutes 4000 bp 2 3 minutes 6000 bp 2 3 minutes Collect DNA from the well using a pipette Proceed to your application using the collected DNA without any further purification You may continue to collect more DNA bands from the same well be sure to fill more water into the second row well or from other wells If your band of interest overruns the collection well and re enters the gel use the REVERSE E Gel program of the iBase device to run the band backwards into the collection well see page 66 amp invitrogen Esa Retrieve DNA from bottom row 65 Collecting DNA Using E Gel CloneWell Agarose Gels continued Run in reverse direction 66 The E Gel iBase Power System is pre programmed with a program to run E Gel agarose gels in a reverse direction ih Toggle between program minutes and seconds by pressing the Mode button until the program blinks Select the REVERSE E Gel Program using the Up Down A V buttons to change the program To change the run time press the Mode button until the minutes or seconds blink and change the values using the Up Down buttons the maximal run time for reverse running is 3 minutes Press the Go button yo start electrophoresis a green light indicates that the run is in progress The LCD displays the count down time while the run is in progress During this period of time monit
127. pre programmed with a SPEED E Gel program for performing runs using high power to generate rapid yes no results The program is suitable for 1 2 and 2 E Gels with SYBR Safe This program is limited to 7 minutes where the bands migrate less than half the length of the gel A run exceeding 7 minutes under these conditions results in a defective run 41 Using E Gel NGS and E Gel with SYBR Safe Gels with the iBase Power System Continued Interruptarunon Electrophoresis can be interrupted at any time by pressing and releasing the Go button to the iBase Power stop the current A flashing red light indicates that the current is stopped and the digital System display flashes the message Press GO to Run Hold Go to Reset to indicate that the run was interrupted You can remove the gel from the iBase device to check the progress of the run then e Continue the run from the point at which it was stopped Reinsert the gel and press and release the Go button The light changes to a steady green and the LCD display shows the count down time The run time but not the program can be adjusted before continuing the run e Cancel the interrupted run Press and hold the Go button for a few seconds The LCD display resets and returns to Ready Mode A new program and run time can be selected to rerun the gel 42 Visualizing E Gel NGS and E Gel with SYBR Safe Agarose Gels Viewing gels containing SYBR Safe gel stain
128. r see below The total volume of the DNA sample and loading buffer should not exceed the volume listed for the second step see table above We recommend using a loading buffer with the following formulation in its final concentration E Gel agarose gels E Gel CloneWell EX and SizeSelect gels e 10 glycerol or 6 e 10 glycerol or 6 Ficoll 400 Ficoll 400 e 10mM Tris HCl pH 7 5 e 1mM EDTA e 0 005 bromophenol blue e 0 005 xylene cyanol FF If using 10X BlueJuice Gel Loading Buffer or TrackIt Loading Buffer page 110 dilute this buffer 50 to 200 fold and add 10 glycerol 117 Two Step Loading of E Gel Agarose Gels Continued Two Step Loading Al wells in the gel must be loaded with either sample or water Avoid introducing Method bubbles while loading as bubbles will cause bands to distort 1 Load deionized water into each well include wells for sample molecular weight marker and empty wells See table on page 117 for volume to load in the first step Do not premix with sample 2 Load 10 pL of sample with loading buffer per sample well 3 Load 10 uL of the appropriate molecular weight markers with loading buffer into the marker well page 49 4 Load 10 pL of water loading buffer may be added into any remaining empty wells High Salt Samples Dilute samples with glycerol loading buffer next page glycerol in deionized water or glycerol in TE buffer to obtain a final glycerol concentration of 10
129. r to remove any excess storage agarose ethidium bromide and plastic from the platform Use a squirt bottle and wipe the platform dry with a clean tissue Do not insert your fingers into the area housing the blades and do not immerse the E Gel Opener in water as the blades may rust Store the E Gel Opener at room temperature 120 Opening E Gel EX and E Gel NGS Agarose Gel Cassettes Introduction The E Gel EX and E Gel NGS agarose gels have a novel openable format that allows the cassette to be opened with a Gel Knife Cat no EI9010 for excision of DNA fragments or for blotting Procedure The following section provides instructions to open an E Gel EX or E Gel NGS cassette Before beginning put on safety goggles and gloves 1 Place the cassette on a flat surface with the wells facing upward 2 Insert the sharp edge of the gel knife a into the groove around the edge of the cassette edge b and lever the knife up and down c C t i 7H in E FE 3 Work around the perimeter of the entire cassette and repeat this action for every edge 121 Opening an E Gel EX and E Gel NGS Agarose Gel Cassettes continued 4 The 4 sides of the cassette should be unsealed Lift off the top of the gel cassette LI E 4 x a 1 v LA P 1 i it M M e 5 Ifyou plan to transfer DNA from the gel by blotting only the main running gel is required Remove the
130. re loaded with RNA loading buffer on the same gel as samples that are loaded with water 53 Visualizing E Gel EX Agarose Gels Viewing E Gel EX agarose gels Imaging E Gel EX agarose gels Important Exposure time and gain setting Disposal of E Gel EX agarose gels 54 E Gel EX gels contain a proprietary DNA gel stain and do not have to be stained after electrophoresis The proprietary DNA gel stain has a fluorescence excitation maxima at 490 nm and an emission maximum at 522 nm when bound to nucleic acids Use a blue light or UV light transilluminator to view the gel a filter is required to photograph the gel your standard ethidium bromide filter may not be appropriate View E Gel EX agarose using these instruments e Blue light transilluminator The E Gel Safe Imager Real time Transilluminator and Safe Imager 2 0 Blue Light Transilluminator Cat nos G6500 and G6600 are compatible for use with E Gel EX agarose gels Refer to the next section for instructions on using the E Gel Safe Imager Real time Transilluminator or Safe Imager 2 0 Blue Light Transilluminator See page 17 for details Blue light transilluminators available from other manufacturers are also compatible for use with E Gel EX agarose gels e Standard 300 nm UV transilluminator e Imaging systems such as laser based scanners equipped with an excitation source in the UV range or between 470 530 nm Note If you plan to excise
131. ress During this period of time monitor the run over a Safe Imager transilluminator When you see the band of your interest migrating into the well press the Go button to stop the run and collect the DNA from the well using a pipette 79 Visualizing E Gel SizeSelect Agarose Gels Viewing E Gel SizeSelect agarose gels Imaging E Gel SizeSelect Agarose Gels Important Exposure Time and Gain Setting 80 E Gel SizeSelect gels contain a proprietary DNA gel stain and do not have to be stained after electrophoresis The proprietary DNA gel stain has a fluorescence excitation maxima at 490 nm and an emission maximum at 522 nm when bound to nucleic acids Use a blue light or UV light transilluminator to view the gel a filter is required to photograph the gel your standard ethidium bromide filter may not be appropriate View E Gel SizeSelect agarose using these instruments Blue light transilluminator The E Gel Safe Imager Real time Trans illuminator and Safe Imager 2 0 Blue Light Transilluminator Cat nos G6500 and G6600 are compatible for use with E Gel SizeSelect agarose gels Refer to the next section for instructions on using the E Gel Safe Imager Real time Transilluminator or Safe Imager 2 0 Blue Light Transilluminator See page 17 for details Blue light transilluminators available from other manufacturers are also compatible for use with E Gel SizeSelect agarose gels Standard 30
132. rm Use a squirt bottle and wipe the platform dry with a clean tissue Do not insert your fingers into the area housing the blades and do not immerse the E Gel Opener in water as the blades may rust Store the E Gel Opener at room temperature The Gel Knife is used to open the cassette for E Gel EX and E Gel NGS agarose gels See page 119 for details on usage Sharpened edge Clean the Gel Knife with mild detergent and water after use and store at room temperature Medium Throughput E Gel Electrophoresis System Medium The system consists of the following components throughput E Gel E Gel 48 gels Each E Gel 48 gel contains 48 sample lanes and 4 marker lanes and is electrophoresis designed for medium throughput agarose electrophoresis of nucleic acids system e E Base Electrophoresis Device The E Base is a base and a power supply all in one device and is an easy to use pre programmed device specifically designed for electrophoresis of E Gel 48 and 96 gels e E Editor 2 02 Software The E Editor 2 02 software allows you to quickly reconfigure digital images of E Gel 48 or 96 gel results for analysis and documentation The E Editor 2 02 software can be downloaded for free from our Website at www lifetechnologies com egels System The Medium Throughput E Gel Electrophoresis System is compatible for use with components multichannel pipettors or automated liquid handling systems The system consists of
133. room may improve visualization The 50 bp ladder see page 111 can be run as a size reference marker Collect the band of interest when the two bands in the 50 bp ladder that bracket the band of interest in size are just beginning to enter for the larger marker and exit for the smaller marker the collection well in the marker lane Run time estimation Band Size Ea Eoen 50 bp 8 5 10 minutes 0 5 1 minute 100 bp 9 10 5 minutes 0 5 1 minute 150 bp 10 11 5 minutes 0 5 1 minute 200 bp 11 12 5 minutes 0 5 1 5 minute 300 bp 12 14 minutes 0 5 1 5 minute 400 bp 13 15 minutes 0 5 1 5 minute 500 bp 14 5 16 5 minutes 0 5 1 5 minute 650 bp 16 18 minutes 1 1 5 minute 800 bp 17 5 19 5 minutes 1 2 minute 1000 bp 18 5 20 5 minutes 1 2 minute The run times indicated are estimates Some bands in different wells may Note migrate differently as DNA fragment size DNA quantity and salt content may slightly affect migration rates 76 Collecting DNA Using E Gel SizeSelect Agarose Gels Introduction After loading your samples proceed with electrophoresis Instructions are provided below to run E Gel SizeSelect agarose gels using the E Gel iBase Power System Electrophoresis 1 Place the amber filter over the iBase device using the iBase Power System 2 Toggle between program minutes and seconds on the iBase device by pressing the Mode button until the program blinks Select the program Run SizeSelect 2 program 8 using the
134. s see page 71 E Gel iBase Power System E Gel iBase Power System The E Gel iBase Power System is an easy to use automated device specifically designed to simplify electrophoresis of single comb or double comb E Gel agarose gels The E Gel iBase is a base and a power supply all in one device The E Gel iBase Power System has an LCD display which shows information about the program selected and running time The display is located near the upper edge of the iBase Just below the display the E Gel iBase Power System has four buttons see image below e A Go button to start programs e A Mode button to toggle between programs minutes and seconds e An Up button marked A to select between programs on the display and increase running time e A Down button marked V to select between programs on the display and decrease running time A LED light is located in the middle of the four buttons which indicates the status of the iBase The gel cassette is inserted into the two electrode connections at the lower half of the iBase At the back the E Gel iBase Power System contains a USB port and a power inlet The supplied power cord has a matching connector that inserts into the power inlet and connects the E Gel iBase Power System to the electrical outlet A separate stand alone power supply is not required to run the iBase The supplied USB cable can be connected to any internet rea
135. s with E Gel Single Comb Gels Introduction 0 8 single comb gel 1 2 single comb gel Results obtained using single comb or double comb E Gel gels are shown below and on the following pages All gels were photographed using a Kodak EDAS120 system You can also use a mini transilluminator to view the bands Note You may vary the amount of markers loaded to improve photography of the gel Results obtained using a 0 8 E Gel gel are shown below using 20 uL per lane Digestion of pUC18 with Pst I lane 5 linearizes the plasmid 2 7 kb Digestion of pcDNA 3 1 5 4 kb with Nco I lane 3 yields 3 fragments 735 bp 1 4 kb and 3 3 kb p Lane Sample 1 High DNA Mass Ladder 200 ng 500 bp DNA Ladder 620 ng peDNA 3 1 Nco I cut 150 ng pcDNA 73 1 uncut 120 ng pUC18 Pst I 60 ng 1 Kb Plus DNA Ladder 300 ng 1 Kb Plus DNA Ladder 300 ng 3 kb PCR fragment 4 kb PCR fragment 5 kb PCR fragment 500 bp DNA Ladder 620 ng High DNA Mass Ladder 200 ng 0 8 Agarose GP 12345 6 T 8 8 igt 12 O COND OFF amp W rn m m C N Results obtained using a 1 2 E Gel gel are shown below using 20 uL per lane Digestion of pUC18 and pcDNA 3 1 are as described above Lane Sample High DNA Mass Ladder 200 ng 250 bp DNA Ladder 400 ng pceDNA 3 1 Nco I cut 150 ng pcDNA 3 1 uncut 120 ng pUC18 Pst I 60 ng 1 Kb Plus DNA Ladder 300 ng 1 Kb Plus DNA Ladder 300 ng 1 kb PCR fragment 2 kb PCR fragment 1
136. se Gels Introduction After you have loaded your samples you are ready to proceed with electrophoresis and retrieve your DNA This consists of three steps 1 Run your fragments to reach the reference line just above the bottom row 2 Run the fragments from the reference line into the bottom well while monitoring the progress constantly 3 Retrieve your fragment from the bottom well Instructions are provided below to run an E Gel CloneWell agarose gel using the E Gel iBase Power System Monitor the E Gel The progress of the E Gel CloneWell agarose gel needs to be monitored during the run CloneWelL with a blue light transilluminator such as the Safe Imager 2 0 Blue Light Transilluminator or E Gel Safe Imager Real time Transilluminator see page 112 Refer to page 67 for instructions on using the Safe Imager 2 0 Blue Light Transilluminator and E Gel Safe Imager Real time Transilluminator Blue light transilluminators available from other manufacturers are also compatible for use with E Gel CloneWell Note Do not use a UV transilluminator since UV light sources could lead to reduced cloning efficiencies Estimated run Refer to the Run Time table below to estimate run times of your fragments to the time to reference Reference Line Same bands in different wells may migrate differently DNA fragment line sizes amounts and salt content may also slightly affect the migration rates The run times indicated are e
137. stead of water you may use a loading buffer to prepare samples or DNA molecular weight marker See page 25 for more details Tracking dye can be used but must be very dilute to avoid masking the bands 71 Molecular Weight Markers DNA molecular We recommend using the following DNA molecular weight markers for different types weight markers of E Gel SizeSelect agarose gels to obtain good resolution Note Supercoiled DNA molecular weight markers may produce a slightly fuzzy pattern when run on E Gel SizeSelect agarose gels E Gel SizeSelect E Gel 1 Kb Plus DNA Ladder 10488 090 Load 100 250 ng 2 agarose 25 bp DNA Ladder 10597 011 markers ina volume of 5 10 uL 50 bp DNA Ladder 10416 014 100 bp DNA Ladder 15628 019 Low DNA Mass Ladder 10068 013 7 2 Using SizeSelect Agarose Gels with the iBase Power System Introduction After preparing your samples proceed with electrophoresis Instructions are provided below to load and run samples on E Gel SizeSelect gels using the E Gel iBase Power System Install the iBase If using only the E Gel iBase Power System attach the power cord with the transformer to the power inlet of the iBase device and plug the other end of the power cord into an Power System l electrical outlet Use only properly grounded AC outlets and cords Install the iBase If using the E Gel iBase Power System in conjunction with the Safe Imager Real time Power System with Transilluminat
138. stimates monitor your gel occasionally during the run Band Size Run time to Reference Line 200 bp 14 18 minutes 400 bp 15 19 minutes 800 bp 17 21 minutes 1000 bp 19 23 minutes 2000 bp 21 25 minutes 3000 bp 24 28 minutes 4000 bp 28 32 minutes 6000 bp 32 36 minutes 63 Collecting DNA Using E Gel CloneWell Agarose Gels continued Electrophoresis of 1 bands to reference line 64 If you haven t already done so place the E Gel iBase Power System over a blue light transilluminator Use the orange cover or orange goggles for viewing the bands For instructions using the Safe Imager transilluminator see page 17 Toggle between program minutes and seconds by pressing the Mode button until the program blinks Select the program Run CloneWell using the Up Down A V buttons Toggle between program minutes and seconds by pressing the Mode button until the minutes blink Enter the estimated run time to the Reference line see previous page using the Up Down buttons Press the Go button on the iBase to run your band of interest to reach the printed reference line just above the bottom row of wells The red light turns to a green light indicating the start of the run Monitor your gel occasionally during the run If your band of interest reaches the reference line press the Go button to stop the run Continue with the next section At the end of the run the iBase stops after the entered ru
139. tep Loading method page 117 Expired gel used Use properly stored gels before the expiration date Longer electrophoresis Longer run times cause an increase in the current resulting in run time or high current poor band migration or melted gel Do not run the gel longer during the run than recommended time for each gel type Sample leaking Sample is overloaded Load the recommended sample volume per well sone WENS Use the Two Step Loading method page 117 Wells damaged during Remove the comb gently without damaging the wells comb removal Failure Mode Defective cassette Disconnect the base and replace gel cassette with a fresh gel continuous rapid cassette Press and release the power button or Go button to beeping and return to Ready Mode Cassette Missing Cold cassette or Use a cassette stored at room temperature Avoid storing gel improper operating cassettes at 4 C Use E Gel iBase PowerBase and conditions E Gel Base at room temperature 20 25 C Hold Go to Reset or a steady red light 36 Electrophoresis of E Gel NGS and E Gel with SYBR Safe Gels Sample Preparation Introduction Materials needed Amount of DNA Total sample volume Preparing samples Loading buffer E Gel NGS and E Gel with SYBR Safe agarose gels contain the safer and environmentally friendly SYBR Safe DNA gel stain enabling visualization of bands with a blue light transilluminator thus minimizing DNA
140. terfering RNA 100 ng well dsRNA diced cut with Dicer enzyme 100 ng well Undiced dsRNA 100 ng well ssDNA 60 mer 200 ng well ssDNA lane 22 annealed to form dsDNA PCR product Hinf I cut PCR product Aat II cut PCR product 40 bp 100 ng well PCR product 72 bp 100 ng well PCR product 150 bp 100 ng well PCR product 240 bp 100 ng well PCR product 317 bp 100 ng well 99 Results with E Gel 96 Gels 2 E Gel 96 with Results obtained using a 2 E Gel 96 with SYBR Safe gel are shown in the figure SYBR Safe below The gel was electrophoresed for 12 minutes using the standard conditions described in this manual and imaged on a Safe Imager 2 0 Blue Light Transilluminator You can vary the amount of markers loaded to improve gel imaging Note The wells of the E Gel 96 gel are staggered DNA bands migrate between adjacent wells in the row below For example the bands of lane A2 will migrate between wells B1 and B2 The box highlights a lane The gel contains the following samples Lane Sample 1 2 11 12 25 ng 10 kb fragment Fermentas Cat no SM1751 3 4 9 10 25 ng 300 ng fragment Fermentas Cat no SM1621 5 6 7 8 25 ng of pUC18 Fermentas Cat no SD0051 2 68 kb cut with EcoR I Cat no 15202 013 M 8 ul of Low Range Quantitative marker Cat no 12373 031 100 Results with E Gel 96 Gels Continued 1 E Gel 96 Gel Results obtained using a 1 E Gel 96 gel are s
141. th Transilluminator the Safe Imager 1 Place the iBase device directly onto the Safe Imager transilluminator so that the legs Transilluminator of the iBase device fit directly into the grooves of the Safe Imager transilluminator 2 Plug the short electrical cord of the Safe Imager transilluminator a into the power inlet of the iBase device b 3 Plug the connecting end of the power cord with the transformer into the back inlet of the Safe Imager transilluminator c and connect the power cord to the electrical socket To power outlet 50 Using E Gel EX Agarose Gels with the iBase Power System Continued Inserta cassette in 1 Open the package and remove the gel Gently remove the comb from the E Gel EX the iBase Power agarose gel using both hands to lift the comb gently by rolling the comb slowly towards you Be careful to pull the comb straight up from both sides Do not bend the comb System Remove any excess fluid using a pipette 2 Slide the cassette into the two electrode connections on the iBase device Press down on the left side of the cassette to secure it into the iBase device The two electrodes on the right side of the gel cassette must be in contact with the two electrode connections on the base The LED produces a steady red light to indicate that the cassette is correctly inserted Slide cassette into electrodes Press left side to secure 3 Load your samples Be sure to load m
142. th and without gel cassette Press and hold the time button Press and release the pwr prg button when no cassette is inserted into the E Base to select the desired program N u Steady green With gel cassette in steady red Without gel cassette no light With gel cassette steady red With gel cassette in steady red Without gel cassette no light No light Last time setting Count down time With gel cassette in last time setting Without gel cassette last program setting Flashing ER EP last program used EP or EG Time increases by 1 minute increments Time increases continuously and automatically stops at 00 Selected program EP or EG Using E Holder Platform Introduction Procedure The E Holder Platform is designed to hold E Gel 48 and96 gels during robotic loading Use the E Holder when you need to load multiple gels on a robotic platform while other gels are running on the E Base Note The E Holder is not a power supply unit cannot be connected to an electrical outlet and cannot be used to run E Gel 96 gels To obtain the best results run E Gel 48 or96 gels on the Mother E Base or Daughter E Base within 15 minutes after loading on E Holder po oY I ow Place the E Holder on the robotic platform Open the package and remove the E Gel 48 or 96 gel Remove comb from the E Gel cassette Place the E Ge
143. the recommended sample volume based on the gel type and loading method For proper band separation we recommend keeping sample volumes uniform Load an equal volume of sample buffer containing the same salt concentration as your sample into the empty wells Gel was not electrophoresed Run the gel within 15 minutes of loading the sample immediately after sample If you cannot run the gel immediately after sample loading loading use the Two Step Loading method page 117 If you are using the E Holder program your robotic system to load the gel 5 minutes before the end of the previous gel s run A1 tip not aligned Be sure to align the A1 tip properly prior to loading your samples on an E Gel 96 gel page 86 Expired gel used Use properly stored gels before the specified expiration date Longer run time or high Longer run times cause an increase in the current resulting current during in poor band migration Do not run the gel longer than the electrophoresis recommended time for each gel type 104 Troubleshooting Continued Uneven run on Differential salt Be sure to load 15 uL of sample buffer containing the same E Gel 48 gels concentration in adjacent salt concentration as the sample into any remaining empty lanes wells Keep all sample volumes uniform Slanted bands in Differential salt Prepare the marker in a buffer containing the same salt marker lanes on concentrations in adjacent concentration as the sa
144. time button If the time button is not released the time setting will increase until it reaches 00 To begin cycling through the numbers again starting from 00 press the time button again Note To increase the run time when a cassette is inserted press and release the time button to increase the time setting by 1 minute intervals or press and hold the time button to increase the time continuously To increase the run time while a run is in progress see next page To manually interrupt or stop a run see page 92 Each E Gel 48 and 96 gel is supplied individually wrapped and ready for use Use short rigid tips for robotic loading To load your samples on the E Holder refer to page 95 for detailed instructions 1 Openthe package and remove the gel 2 Remove the plastic comb from the gel 3 Slide the gel into the two electrode connections on the Mother E Base or Daughter E Base The two copper electrodes on the right side of the gel cassette must be in contact with the two electrode connections on the base as shown below 4 When the gel is properly inserted into the base a fan in the base will begin to run and a red light will illuminate at the lower left corner of the base The digital display will show the appropriate time for a selected program or last time setting Ready Mode _ _ Electrode _ Electrode Note If you accidentally inserted a gel into the base before selecting program EG remove the
145. to select the appropriate program for your gel Gel Type Program Default Run Maximal Run Time Time E Gel with SYBR Safe RUN E Gel 0 8 2 0 26 minutes 40 minutes E Gel NGS RUN E Gel 26 minutes 32 minutes The SPEED E Gel program is available for E Gel with SYBR Safe gels see page 32 The default run time for the RUN E Gel program is 26 minutes To change the run time press the Mode button until the minutes or seconds blink Use the Up Down buttons to change the values up to the maximal run time Press the Go button to start electrophoresis a green light indicates that the run is in progress The LCD displays the count down time while the run is in progress The device stops automatically when the programmed time has elapsed A flashing red light and beeping rapid beeping for 30 seconds followed by a single beep every minute signals the end of the run The LCD displays Run Complete Press Go Press and release the Go button to stop the beeping The LED shows a steady red light and the LCD display shows the most recent program and settings Remove the E Gel cassette from the iBase device You are now ready to proceed to imaging or any other application with the gel e To open the E Gel with SYBR Safe cassette for excision of DNA fragments or for blotting see page 119 for details e To open the E Gel NGS cassette for excision of DNA fragments or for blotting see page 119 for details The iBase device is
146. tte inserted into a base Press and release the pwr prg button Automatic Automatic Press and release the pwr prg button during the run Continuous beeping for 2 minutes followed by a single beep every minute Continuous beeping for 2 minutes followed by a single beep every minute No light if a cassette is not inserted or red light if a cassette is inserted Steady red Steady green Flashing red until the time button is pressed Alternating red and green With gel cassette in steady red Without gel cassette no light Without gel cassette EP last program used EP or EG With gel cassette in last time setting Default time setting 12 minutes for EG 14 minutes for EP or last time setting Count down time Negative time display 00 to 19 minutes Negative time display 00 to 19 minutes Flashing time display 93 E Base Quick Reference Guide Continued Mode Action Sound Light Digital Display Return to Ready mode after an automatic stop Restart after a manual stop Return to Ready mode after a manual stop Failure No cassette Time setting Program setting Press and release the pwr prg button Press and release the pwr prg button Press and hold the pwr prg button Press and hold pwr prg button for 2 seconds and remove gel from the base With gel cassette in Press and release the time button Wi
147. ualification Analysis information for each product Certificates of Analysis are available on our website Go to www lifetechnologies com support and search for the Certificate of Analysis by product lot number which is printed on the box Limited product Life Technologies Corporation and or its affiliate s warrant their products as set forth in warranty the Life Technologies General Terms and Conditions of Sale found on Life Technologies website at www lifetechnologies com termsandconditions If you have any questions please contact Life Technologies at www lifetechnologies com support 113 Appendix B Safety Explanation of Symbols and Warnings E Base CE u LISTED E189045 Caution A z WEEE m Double Insulation 114 The Mother E Base and Daughter E Base comply with the Underwriters Laboratories Inc regulation and the European Community Safety requirements Operation of the E Gel bases is subject to the following conditions Indoor use Altitude below 2 000 meters Temperature range 5 to 40 C Maximum relative humidity 80 Installation categories over voltage categories II Pollution degree 2 Mains supply voltage fluctuations not to exceed 10 of the nominal voltage 100 240V 50 60Hz 1500 mA The Mother E Base has been tested with up to 3 Daughter E Bases connected at one time Mains plug is a disconnect device and must be easily accessible Do not attempt to open E Bas
148. verexposed To ensure that the barcode label is distinct and readable in the image experiment with different shutter settings for your particular camera 87 Loading E Gel 48and 96 Gels Continued the gel from the plastic pouch and run the gel within 15 minutes of loading If a gel has been out of its plastic pouch for more than 15 minutes you must use the Two Step Loading method described on page 117 e Do not pre run E Gel 48 and 96 gels e Store and run E Gel agarose gels at room temperature e For optimal results load each E Gel 48 and 96 gel within 15 minutes of removing Important Selecting Program The recommended program for E Gel is EG and the run time for E Gel 48 196 and 296 gels is 20 minutes E Gel 48 4 is 17 minutes and E Gel 96 gels is 12 minutes Alternatively E Gel 96 gels can be run using the EP program with a 6 minute run time though in some cases this may result in a slight reduction of run quality on E Base You will need to select an appropriate program on the base prior to inserting a gel into the base Note If you had previously set the E Base to the desired program or set the time the last used program or time is displayed 1 Plug the Mother E Base into an electrical outlet using the plug on the base If using Daughter E Base connect the Daughter E Base to a Mother E Base or to another Daughter E Base connected to a Mother E Base 2 The display will show EP default
149. wer supply unit cannot be connected to an electrical outlet and cannot be used to electrophorese E Gel agarose gels 111 Accessory Products Continued Gel Knife E Gel Opener E Editor 2 02 Software Safe Imager Transilluminators Loading Buffers 112 The gel knife Cat no EI9010 is used to open E Gel EX cassettes for excision of DNA fragments or blotting The E Gel Opener is a device specifically designed to open E Gel single comb and double comb cassettes excluding E Gel EX cassettes for excision of DNA fragments or blotting Ordering information is provided below Product Quantity Catalog no E Gel Opener 1 G5300 01 E Gel Opener Replacement Blades 10 G5350 10 The E Editor 2 02 software is available FREE of charge with the purchase of any E Gel 48 or 96 gels and related equipment The software may be downloaded at www lifetechnologies com egels The E Gel Safe Imager Real time Transilluminator and Safe Imager 2 0 Blue Light Transilluminator are specifically designed for use with E Gel EX E Gel SizeSelect and SYBR Safe stained DNA gels See pages 125 126 for viewing options Product Quantity Catalog no E Gel Safe Imager Real time Transilluminator 1 G6500 Device and Amber Filter TM amp TM G6465 E Gel iBase and E Gel Safe Imager Combo Kit a 1kit G6465EU id G6465UK E Gel Safe Imager Real time Transilluminator parce Starter Kit for Cloning US EU
150. with the two electrode connections on the base The LED produces a steady red light to indicate that the cassette is correctly inserted Slide cassette into electrodes Press left side to secure 4 Take out the comb and load your samples Be sure to load molecular weight markers and add water to any empty wells Note It is not necessary to pre run E Gel single comb or double comb agarose gels Load the E Gel E Gel agarose gels are designed for loading samples manually or using a multichannel agarose gel pipettor We recommend the following methods of sample loading based on the gel type Gel Type Method of Loading E Gel single comb Manual E Gel double comb Manual or multichannel pipettor 30 Using E Gel Agarose Gels with the iBase Power System continued One step loading method Electrophoresis using the iBase Power System Load the E Gel agarose gel within 15 minutes of opening the pouch and run the gel within 1 minute of loading samples Avoid introducing bubbles while loading as bubbles will cause bands to distort 1 Remove the comb from the E Gel gel using both hands to lift the comb gently by rolling the comb slowly towards you Be careful to pull the comb straight up from both sides Do not bend the comb Remove any excess fluid using a pipette Load samples in 20 uL volume into the wells Load 20 uL of water into any remaining empty wells Load 100 250 ng of the appropriate molecular weight markers
151. y does not generate liquid waste e SYBR Safe DNA gel stain does not induce transformations in primary cultures of Syrian hamster embryo SHE cells In contrast ethidium bromide tests positive in the SHE cell assay consistent with its known activity as a strong mutagen e SYBR Safe stain does not cause mutations in mouse lymphoma cells at the TK locus nor does it induce chromosomal aberrations in cultured human peripheral blood lymphocytes with or without S9 metabolic activation e Compared to ethidium bromide SYBR Safe DNA gel stain causes fewer mutations in the standard Ames test Weakly positive results occurred in only four out of seven Salmonella strains and only with activation by a mammalian S9 fraction e Asingle oral administration of SYBR Safe DNA gel stain produces no signs of mortality or toxicity at a limit dose of 5000 mg kg View studies documenting the safety of SYBR Safe in the SYBR Safe White Paper document available from www lifetechnologies com content dam LifeTech global life sciences pdfs 494 pdf SYBR Safe DNA gel stain is not classified as hazardous waste but because disposal regulations vary please contact your safety office or local municipality for appropriate SYBR Safe disposal in your community SYBR Safe DNA Gel Stain Continued Spectrum of SYBR Bound to nucleic acids SYBR Safe stain has fluorescence excitation maxima at 280 and Safe 502 nm and an emission maximum at 530 nm see
152. your DNA UV light sources can lead to reduced cloning efficiencies Using a blue light transilluminator will also minimize your personal UV exposure Photograph E Gel NGS and E Gel with SYBR Safe gels using a CCD camera or a laser based scanner For photographing gels refer to page 127 to determine the optimal filter sets to use or contact the instrument manufacturer for advice Do not use ethidium bromide filters that block light above 500 nm for photographing E Gel with SYBR Safe The band intensity of E Gel NGS agarose gels can be improved by incubating the gel at 4 C for 5 minutes prior to imaging While yielding similar sensitivities to ethidium bromide SYBR Safe gel stain is somewhat dimmer yet with a lower background than ethidium bromide As a result a slightly longer exposure time or higher gain setting may be necessary SYBR Safe gel stain shows no or very low mutagenic activity when tested by an independent licensed testing laboratory and is not classified as hazardous waste under US Federal regulations As disposal regulations vary please contact your safety office or local municipality for appropriate SYBR Safe gel stain disposal in your community 43 Results using E Gel with SYBR Safe Agarose Gels Introduction E Gel 1 2 with SYBR Safe E Gel 2 with SYBR Safe 44 On this page we display typical results using E Gel with SYBR Safe agarose gels 1 2 and 2 On the next page exa
153. zeSelect Agarose Gels with the iBase Power System ccscsssssessseseeeseseeeeeseseseseseacaeseseeeeeeeeeeteeseaees 73 Run Time Estimation for SizeSelect Agarose Gels cue mca oie teda lle nude dea td edet t dde tus 76 Collecting DNA Using E Gel SizeSelect Agarose Gels 77 Collecting DNA Using E Gel SizeSelect Agarose Gels Continued sss 78 Visualizing E Gel 9izeSelect M Agarose Gels ette ee deo ip tete elei ip comes ted Pes ie acida 80 Quantitation of DNA Isolated from E Gel SizeSelect Agarose Gels eene 81 TOU LSS NOON uM DM m 82 Electrophoresis of E Gel 48 96 Gels sccsssssessseessessnsnseceeesensnneeceessensnneeceesesensnnecceessessnnecoeessensaecsooess 84 General Guidelines for E Gel 49 96 Gels uestis vor a vto Ed o Dec e E HR PIN Due nema endows 84 Loadins E Gel 45 and 96 Gels cuisse osa A dut ute E Sc I IN UNUS d D E OD 87 Ruine Gel 8 a 96 Ge a edit o MM tide Menu note eats dyes uppa dtp ot pape USo iea 91 IE PE Nt Ock Rererence INO RR 93 Wisi Eller lo Motte eracct setae tie ce re E ce ec ee eats 95 Visualizing E Gel 96 with SY BR Safe Agarose Gels 5 cid east ta eaey sees deh cae alte bubulo E edite totales eus 96 RESE N N E to I C m MM MT 97 Results wat dale e qu e mee 100 sine EsEdit or2 02 50f Wale sisse dents oie tva datas dene edic tetas cea Saat estu tales pide ba chu eina tou end 103 Troubleshooting miara MM 104 1V ADDODUODadiemiciniak estu mi ceniuis nte idu

E-Gel® Technical Guide - Thermo Fisher Scientific

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E-Gel&reg; Technical Guide - Thermo Fisher Scientific

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E-Gel® Technical Guide - Thermo Fisher Scientific