D4 Horizonal Electrophoresis Systems User Manual
Contents
1. Warranty Information Section 10 Gaus Lsioad L006 OSI CHG O Nad UONEWJOJU A UBJEM JO JOJNQUISIP E90 INOA DEDUOO WSN By ap s no suoneai dde jeloads pue sones eoueuajuleW uonejedo Aueuem Juswd nba uo suonsanb noA Jamsug 0 Apeal 919M 29 4 8 2 0v L JO epeueo JO YSN 198 267 008 1 le juauyuedag sexies jedIUYOE INOA peo eseajd Pasinbai si sones JUSWdINbe y ae9ueuajuiew aMmjuanald pue uoneJado UOHE e SU juswdinba ikjap AjjnjaJeo sIenuew UO ONI SU paud saAllle juswdinba InoA aJjojaq uoNeunojum voneJedald aus 2AIsuaya dWoo UM djey 0 Apea s 221 0Q sejes oWJaY B20 INOA s onpoud Jo sso JO s yoJd s0 0 sabewep uonejiwi joyym Buipnjou sabBewep jenuanbasuoo Jo papu Aue Joj a qei aq jou jjeys oway AlddV TIYHS 3SOdYNd YYINOIILYYVd Y YOA SSANLIA YO ALITAVLNVHOYAN AO SSILNVYYVM ON Gal Ida YO WHO NALLIYM YIHLIHM SSILNVYYVM Y3H LO TY SO NAIN NI ANY SAISNIOXS SI ALNVYYYM SIHL LOoNeuNssp goy peddiys se syed juewssejdal pue pied aBejsod oway o peuinjes aq snw syed BulWjojuoo Uou je uondo sowy y USWdINbS Jo juauoduoo9 Aug jo uime JO enoldde Jod anib snu Juswuedagq sesiales JedIUYOS ay pollad Alueuem jeUIBUO ay PUCASG Jed JUSUOdLUOD ay 0 JO juaudinba ay Jayya o AIUEUEA BY puajxe zou jeys JUeJem siy Japun Juswd nba JO sped Jusuodwos jo Jeda JO juswase day AJUBLIEM SIY wo papnj9xe ose sjoxseb pue say ssejB sway ajgepuadx3 Spa Aue jo aoueuuo J9d O2. JOUd UON DAJIp pue UONEUIUE SP AE UE Jo pajoejuo9 aq Jsnw Juswpedag sexies peoluyoa oul juswaaibe AjueJem siy Aq pala nod zou SI uoe pue uoheiqijeo uonejje suj oqe Bundaoxa esuadxa s OWJBY Je paveda aq IIm diysueunJom JO IEUSIEUU Ul Bulwjojuoo uou aq o USAOJd sued juaeuodulos syjuow 9 Xis Au 38114 ey Hung J9uMO juanbasqns Aue o spua xa uor 98 01d JUEJemM ay palaailap si Juswd nba noA aun wes ay jejewixoldde ye joaye ou 0b im jueJem ay os aun Buiddius smojje siy AuIpDEL ano OU paddiys si juaudinba noA app ay vo syjuow OM SUEIS pollag AIUEUEAA SUL TIWNOILYNYSLNI ALNVYYVM SLINAOYd IMO SISILNSIDS 43HSIA ONY HL Thermo Scientific Horizontal Electrophoresis 10 2 Thermo Fisher Scientific 401 Millcreek Road Marietta Ohio 45750 United States www thermofisher com ThermoFisher SCIENTIFIC
3. Jo Pal pu Aue Jo a qei aq Jou jeys oway AlddV TIVHS ASOdYNd YVINOILYVd Y 404 SSINLIA YO ALITAVLNYHOYAN AO SIILNYYYYM ON CallId YO IYYO NILLI4M YAHLIHM S3ILNVYYYM Y3HLO T1V 40 NAIN NI ANY SAISN1OX4 SI ALNVYYVM SIHL uoreuNssp g04 paddiys ag syed Juswaseida4 pue pied aBejsod oway 0 pauinjai aq Jsnw syed BuiwJojuod uou e uondo s owlay 1y Juswd nba Jo jusuodwoo Aue jo unya Jo enodde 1oud anib snw juauuedag sexinas EIIUY99L ay poled AupUpw eui6iJo ay puoAaq Jed JUSUOGWWOD y 0 Jo juaudinba ay Joe O uenem sy puajxe JOU jeys jueJem siy Jopun uswdinba JO syed jusuodwoo JO Jeda JO juswa2g day AJUCIIEM SIY W01 papn oxa ase sjayseb pue sia ssej6 sway ajgepuadxy sed Aue jo adue WJojJad O JOId UOIOSJIP pue UONEUIUSISP AJUBUEM Jo pajoejuo9 aq Jsnw jUsWPedEG sexies IEOIUUDel ay Juswaaibe AjueJem siy Aq pes8Aod zou SI uoe pue uoleqijes uonejje suj Joqe Hulpnjou esuadxa s oWJay Je paoejdau aq m d ysuewy Jom JO ella gw Ul BUIWIIOJUOO UOU aq 0 UBAOJd syed jusuodwoo syjuow gE xIS AjJIU 1814 ayy Bulung Jaumo juanbasqns Aue o spu x uonoajold AUEUEMW ay patanijop si Juswd nba noA aun swes ay jejeuixoldde Je JOSE ojul 06 JIM JUeJJem ay os aun Buiddiys smojje siy AE Ino woy paddiys si juaudinba Jno ajep y WO syaaMm OM SUEIS pollad AIUSUEM SUL VSN ALNVYYVM SLONGOYd IMO SISILNSIDS Y3IHSIA OINYIHL 10 1 Horizontal Electrophoresis Thermo Scientific
4. the order or catalog number on your invoice and check the corresponding part lists Description E aa p pem Dein ern EN a paan rare Ga E pa jm _ Comb 1 5mm thick 17 tooth includes one marker lane e Horizontal Electrophoresis 3 1 Section 3 Unpack and Check Your Order Table 3 1 Specifications and Recommended Running Conditions for Model D4 16 x 17 1 Voltage requirements V 3 2 Horizontal Electrophoresis Thermo Scientific Section 4 Setting Up There are two casting options with the D4 system The first option is Casting Two Gels simultaneously in the external casting chamber The second option is Casting One Gel within the buffer chamber e 16 4cm R There are two comb options available with the D4 Gel Tray The first option allows 17 samples 5 2cm 5 2cm 5 2cm with a 16 4cm run length or the second option lt kaan allows 51 samples with a 5 2cm run length i Figure 4 1 Figure 4 1 Combs Casting Two Gels pa Remove the SuperSafe lid from the gel box by holding the front of the buffer chamber and pulling the lid off by holding the center of the back of the lid or pressing your thumbs on both sides of the lid The SuperSafe lid is attached to the unit at the connection of the power cords to the banana plugs 2 For shipping and convenient storage the gel trays are packaged inside the casting chamber Figure 22 4 2 To remove the gel trays hold IG the casting chamber fir
5. Horizontal Electrophoresis System Model D4 Operating and Maintenance Manual 7007325 Rev 0 Visit us online to register your warranty www thermoscientific com warranty Gi Owl 5 Preface MANUAL NUMBER 7007325 0 4 9 12 Transferred to Marietta was Rev Date 11 2002 CCS REV ECR ECN DATE DESCRIPTION By Thermo Scientific Horizontal Electrophoresis i Preface CAUTION Contains Parts and Assemblies Susceptible to Damage by Electrostatic Discharge ESD Important Read this instruction manual Failure to read understand and follow the instructions in this manual may result in damage to the unit injury to operating personnel and poor equipment performance A Caution All internal adjustments and maintenance must be performed by qualified service personnel A Material in this manual is for information purposes only The contents and the product it describes are subject to change without notice Thermo Fisher Scientific makes no representations or warranties with respect to this manual In no event shall Thermo be held liable for any damages direct or incidental arising out of or related to the use of this manual 2012 Thermo Fisher Scientific All rights reserved Horizontal Electrophoresis Thermo Scientific Preface Important operating and or maintenance instructions Read the accompanying text carefully gt gt gt Potential electrical hazards Only qualified persons should perform proced
6. ate port inserts packaged in a small plastic bag located inside the unit upon arrival How these work The inserts are pushed into the attached ports on the side wall of the unit with the black O ring side facing in The insert will snap into place in the port in the open position and is ready to circulate buffer Appropriate tubing is then connected to the small outer ringed ends of the ports for circulation using a separate recirculator or peristaltic pump To close the port which also releases the insert simply press the flat metal button and the insert detaches The port is now in the closed position Note Buffer may also be passed through a heat exchanger A Horizontal Electrophoresis 9 1 Section 10 Warranty Information OE ETRE L006 OSI ch 6 0 ASN UONEWJOJU AJUCLIEM JO J0 nq isip 8901 INOA DEIDUO YSN 24 APISINO suonesidde elo ads pue ous soueuajuiew uollelsdo Kyueulem juswdinba uo suonsanb moi Jamsue o peal 919M 29 8 8 07 L Jo epeueo pue VSN 1984 864 008 L Je juauyedag sadi nag EIIUy99L INOA jes aseajd pa nba si B21 Alas Juawd nba y as9ueuajulew SAI UBAVId pue uonejado uone je sui juawdinba 1ejap Ajjnjaseo sjenueu UODOPUISUI pajulid SOALIE juswdinba noA ssojeq UONeWIOJU uoneledaid ous aarsuayalduos YUM djey o Apea s SOO sejes oW1aY 290 INOA s onpold JO sso JO S iJOud sot 0 saBewep uongjiwi noyym Hulpnjoul sabewep enuenbasuos
7. e UVT gel tray s If numerous gels are to be run in one day a large volume of gel solution may be prepared and placed in a covered bottle stored between 40 60 F in a water bath This provides a ready gel supply in a warm liquid form that will solidify quickly when gels are cast Figure 4 3 Pour Pipette Agarose 5 Pour or pipette Figure 4 3 the appropriate amount see Table 4 2 of warm agarose lt 60 F onto the UVT gel tray that has been placed into the casting position in the casting chamber Immediately after pouring insert the desired comb or combs into the comb slots to form the sample wells Repeat with second gel tray if casting 2 gels Allow the gel s to solidify completely If a short running distance is required for proper sample separation then 3 combs may be used This expands the number of samples per run 4 2 Horizontal Electrophoresis Thermo Scientific Section 4 Setting Up Table 4 1 Mobility range of DNA in different percentage agarose gels Agarose w v Table 4 2 Amount of Agarose to prepare Gel volume is determined by the following formula and may be adjusted according to need or preference gel width cm X gel length cm X gel thickness cm ml of agarose Width of Gel cm Length of Gel cm Thickness of Gel cm Volume of Gel ml Table 4 3 Sample Volume D4 unit volumes of comb wells Thickness Width of Bi of Teeth Recommended loading volume ul For different t
8. e running at an angle level on the casting chamber platform the bubble in the bubble level should rest in the center circle Adjust the leveling screws to make the casting chamber D4 CST level Check to be sure the platinum electrode wire is intact and running evenly across the base of the chamber and up the side to the junction of the banana plug If there appears to be a break in the electrode connection contact Technical Services Samples seem to be running unevenly in certain areas immediately This problem may also be caused by regular casting with very hot agarose gel gt 60 F which may damage the gel tray over time Always cool the melted agarose to below 60 F before casting to avoid warping the UVT gel tray Warping the gel tray will cause all subsequent gels to be cast unevenly Gels should be no more than 5mm thick and allowed to solidify completely before running For standard agarose this would be about 30 minutes if low melting point agarose is used it may be necessary to completely solidify gels at a cooler temperature in the refrigerator or cold room Gels should be submerged in 3 5mm of buffer to avoid gel dry out but excess buffer gt 5mm can cause decreased DNA mobility and band distortion Samples do not band sharply and appear diffuse in the gel Check to be sure that a complete power circuit is achieved between the unit and the power supply Platinum wire and banana plugs should be intact To test sim ply fill
9. e seals or other accessories are required The model D4 s design allows you to run one gel tray 51 samples or two gel trays 102 samples while saving valuable bench space Each of the 2 UVT Ultra Violet Transmissible gel trays 16cmWx 17cmL accommodates up to 3 combs allowing the user to run up to 3 series of samples of equal distances A stand alone casting platform is included for casting 2 gels simultaneously A single gel can be cast right in the buffer chamber Custom combs are available upon request Horizontal Electrophoresis 1 1 Thermo Scientific Section 2 Safety Information Warning Please read carefully before operating A e To avoid the risk of personal shock always disconnect the gel box from the power supply Further the power supply must be equipped with a shutdown on disconnect circuit e Statement of Proper Use Use this product only for its intended purpose as described in this manual Do not use this product if the power leads are damaged or if any of its surfaces are cracked e Running conditions for this unit should not exceed the name plate readings found on the lower buffer chamber e Do not move the unit unless the power source to the unit has been disconnected Horizontal Electrophoresis 2 1 Thermo Scientific Section 3 Unpack and Check Your Order Before starting unpack the unit and inventory your order If any parts are missing contact Technical Services within 48 hours Reference
10. en Care in handling the powder and stock solution must be taken Always wear gloves when handling the powder solutions and all gels that contain ethidium bromide 7 2 Horizontal Electrophoresis Thermo Scientific Thermo Scientific Section 7 Reagents Information Table 7 3 Preparation and Properties of TAE and TBE Electrophoresis Buffer Systems These buffers are used because they both have a basic pH which gives the phosphate group of the DNA a net negative charge allowing migration of the DNA toward the positive anode in the electrophoresis chamber TAE Tris Acetate with EDTA 40mM Tris Base 40mM Acetic Acid 1mM EDTA ESTO ES 1 TBE Tris Borate with EDTA 89mM Tris Base 89mM Boric Acid 2mM EDTA CI Pen I Horizontal Electrophoresis 7 3 Section 7 Reagents Information 7 4 Horizontal Electrophoresis Choose the buffer best suited to the experiment Each buffer has different properties providing the necessary ions for electophoretic migration Buffer Suggested Use TAE Buffer e Use when DNA is to be recovered e For electrophoresis of large 520kb DNA e Applications requiring high resolution e Has low ionic strength and low buffering capacity recirculation may be necessary for long runs gt 4hrs TBE Buffer e General Purpose Buffer e Can be re used e For electrophoresis of small lt 1kb DNA e Better resolution of small lt 1kb DNA e Decreased DNA mobility e High ionic strength and high buffering capac
11. er is added the mobility of the DNA decreases and band distortion may result Excess buffer causes heat to build up and buffer condensation inside the unit may result The gel seems to run slower under usual running conditions Additional Sources for Reference Maniatis T E E Fritsch and J Sambrook Molecular Cloning A Laboratory Manual Cold Spring Harbor Laboratory Cold Spring Harbor NY Short Protocols in Molecular Biology A Compendium of Methods from Current Protocols in Molecular Biology Edited by Fredrick M Ausubel et al Adams D and R Ogden Electrophoresis in Agarose and Acrylamide Gels Methods in Enzymology Vol 152 1987 Academic Press Inc Fotador U Simultaneous Use of Standard and Low Melting Agarose for the Separation and Isolation of DNA by Electrophoresis Bio Techniques Vol 10 No 2 1991 Boots S Gel Electrophoresis of DNA Analytical Chemistry Vol 61 No 8 April 15 1989 8 2 Horizontal Electrophoresis Thermo Scientific Thermo Scientific Section 9 Optional Equipment Buffer Exchange Port Option D4 BP The buffer exchange port option is used to recirculate the buffer during extended gel runs Recirculation is used to prevent buffer depletion of certain low ionic running buffers for extended runs multiple sample sets or for RNA gels If your unit has the buffer exchange port option it will be fitted with two white buffer port terminals Figure 12 and will contain two separ
12. hicknesses of gel Thermo Scientific Horizontal Electrophoresis 4 3 Section 4 Setting Up 4 4 Casting One Gel D One 1 gel may be cast directly in the buffer chamber of the device 1 Figure 4 4 Casting Position Horizontal Electrophoresis Remove the SuperSafe lid from the gel box by holding the front of the buffer chamber and pulling the lid off by holding the center of the back of the lid or pressing your thumbs on both sides of the lid The SuperSafe lid is attached to the unit at the connection of the power cords to the banana plugs Place the UVT gel tray into the buffer chamber in the casting position Figure 4 4 Immediately after pouring the appropriate amount of agarose lt 60 F onto the UVT gel tray insert the desired comb or combs into the comb slots to form the sample wells Once gel is ready to run place the tray in the running position Figure 4 5 b Figure 4 5 Running Position 3 Preparing the gel s The percentage of agarose and the buffer used is determined by the size of the samples to be separated and further recovery of the samples see Tables 4 1 4 2 and 4 3 The agarose and buffer are mixed and heated over a heat source in a microwave oven or in an autoclave until the agarose is completely dissolved The prepared gel solution then must be cooled to below 60 F before casting to avoid warping the UVT gel tray s If numerous gels are to be run in one day a large volume
13. ity recirculation may not be required for extended run times e Reacts with the agarose making smaller pores and a tighter matrix This reduces broadening of the DNA bands for sharper resolution 3 Sample Buffer Samples are prepared and mixed with sample buffer before being applied to the prepared gel Sample buffers contain similar components to the running buffer dyes for visibility and glycerol to provide weight to the samples This increased sample density ensures samples load evenly into the wells and do not float out during loading Dyes also migrate toward the anode end of the electrophoresis chamber at predictable rates allowing the gel run to be monitored 4 DNA Markers Markers are run on each gel to monitor sample separation and to provide an accurate size estimation of the samples By running a known marker of a specific concentration the amount of the DNA can be estimated These size markers are a suitable restriction digest of commonly available DNA Thermo Scientific Section 8 Troubleshooting Check to see if the gasket is firmly seated in the grooves on the ends of the UVT Agarose leaks into chamber when pouring gel gel tray Reseat gasket if necessary by removing and rinsing under warm running water then reseat evenly in the tray groove Check to be sure the casting is being done on a level surface A leveling platform may be required Make sure the gel tray is pressed all the way down and rests Bands seem to b
14. l Note An increased agarose provides better separation of small fragments and bands very close together that tend to be more difficult to separate A specialty agarose product formulated to increase resolution of low molecular mass samples may also be used or an agarose additive may be added to standard or low melting point agarose Example A good mid range gel percentage would be 0 7 or 0 7g agarose in 100ml electrophoresis buffer TBE or TAE following heating and dissolving the agarose 10ul of ethidium bromide stock solution Smg ml is added The gel would be run with compatible electrophoretic running buffer 1X TBE or 1X TAE that also contained ethidium bromide One liter of the running buffer would contain 100ul of this 5mg ml ethidium bromide stock solution 2 Ethidium Bromide For photodocumentation of samples the gel may be stained during or following the run with a variety of stains The most common stain for DNA is ethidium bromide Ethidium bromide may be added directly to the gel and running buffer to visualize and photograph the separated fragments following the gel run without the need for an additonal staining step The ethidium bromide is added to both the gel after heating and the electrophoresis buffer at a concentration of 0 5ug ml Conversely the gel may be stained in a concentrated ethidium bromide solution after the gel run and rinsed for visualization Warning Ethidium bromide is a potential carcinog
15. mly with one hand grasp the long sides of the UVT gel tray and pull up slowly at an angle with your other AY hand The trays fit snugly for leak proof gel casting therefore they Figure 4 2 Casting Gels may be tight Walking the tray upwards at an angle may be helpful The tightness will diminish the more the unit is used 3 To cast two gels place the gel trays into the casting chamber Figure 4 2 so the gasketed ends press against the walls of the casting chamber Make sure the gel tray is pressed all the way down and rests level on the platform The bubble in the bubble level should rest in the center circle To level the casting chamber adjust the leveling screws on the front of the casting chamber the third screw is for balance until the bubble is in the center circle Thermo Scientific Horizontal Electrophoresis 4 1 Section 4 Setting Up Casting Two Gels 4 Preparing the gel s The ntin percentage of agarose and the co ued buffer used is determined by the size of the samples to be separated and further recovery DES A ns of the samples see Tables 4 1 L t 4 2 and 4 3 The agarose and Py buffer are mixed and heated S a aa Apk L over a heat source in a microwave oven or in an ge e autoclave until the agarose is completely dissolved The prepared gel solution then must be cooled to below 60 F before casting to avoid warping th
16. n is complete and tracking dye has migrated as far through the gel as desired or to the end of the gel turn off the power supply and slide off the lid to disconnect the unit from the power source Carefully remove the tray s containing the gel wear gloves if ethidium bromide is present The UVT gel tray makes visualization and photography with a UV light source easy without the need to remove the gel from the tray Thermo Scientific Thermo Scientific Section6 Care and Cleaning Caution Do not use ethanol or other organic solvents to clean Owl products Organic solvents cause acrylic to craze or crack Clean all Owl acrylic systems with warm water and a mild detergent Do not autoclave bake or microwave your unit Temperatures over 50 C can do damage to the acrylic A The unit may be rinsed with warm water or cleaned with warm water and a mild detergent to get rid of any debris Note If an RNase free electrophoresis system is desired there are various methods to rid the system of RNA contamination For fast and easy decontamination use RNase Away Spray wipe or soak labware with RNase Away then wipe or rinse the surface clean it instantly eliminates RNase RNase Away eliminates the old methods that include treatment with 0 1 Diethyl Pyrocarbonate DEPC treated water and soaking in dilute bleach DEPC is suspected to be a carcinogen and should be handled with care This electrophoresis system should never be autocla
17. nit to be repaired at our expense and to your satisfaction Regardless of your needs our professional telephone technicians are available to assist you Monday through Friday from 8 00 a m to 6 00 p m Eastern Time Please contact us by telephone or fax If you wish to write our mailing address is Thermo Fisher Scientific 401 Millcreek Road Box 649 Marietta OH 45750 International customers please contact your local Thermo Scientific distributor v Horizontal Electrophoresis Thermo Scientific Thermo Scientific Section 1 Section 2 Section 3 Section 4 Section 5 Section 6 Section 7 Section 8 Section 9 Table of Contents Introduction Zeg SE ee near 1 1 Safety Information u ea 2 1 Unpack and Check Your Order z22220s sen nennen 3 1 Casting Two Gelso id apn KA 4 1 Setting eet ee 4 1 Casting Two Ge da edel 4 1 Casting One Gel 22 a EE 4 4 Using the System 5 1 Finishing Up 3422242427442 2 Hecke ieh 5 4 Care and Cleaning 0cc cee cece eect eee eeeeeaeees 6 1 Reagents Information 7 1 Troubleshooting 00 cece eee eee eee eee ees 8 1 Optional Equipment 00 cece eee eee eee eee 9 1 Horizontal Electrophoresis V Thermo Scientific Section 1 Introduction The Owl Horizontal Agarose Gel Electrophoresis System is designed to provide flat even banding patterns and consistent results with hassle free gel casting No tape grease agaros
18. o 6 00 p m Eastern Time at 1 740 373 4763 Direct 1 800 438 4851 Toll Free U S and Canada 1 877 213 8051 FAX http www thermoscientific com Internet Worldwide Web Home Page service led marietta thermofisher com Tech Support Email Address www unitylabservices com Certified Service Web Page Our Sales Support staff can provide information on pricing and give you quotations We can take your order and provide delivery information on major equipment items or make arrangements to have your local sales representative contact you Our products are listed on the Internet and we can be contacted through our Internet home page Our Service Support staff can supply technical information about proper setup operation or troubleshooting of your equipment We can fill your needs for spare or replacement parts or provide you with on site service We can also provide you with a quotation on our Extended Warranty for your Thermo Scientific products Whatever Thermo Scientific products you need or use we will be happy to discuss your applications If you are experiencing technical problems working together we will help you locate the problem and chances are correct it yourself over the telephone without a service call When more extensive service is necessary we will assist you with direct factory trained technicians or a qualified service organization for on the spot repair If your service need is covered by the warranty we will arrange for the u
19. of gel solution may be prepared and placed in a covered bottle stored between 40 60 F in a water bath This provides a ready gel supply in a warm liquid form that will solidify quickly when gels are cast Pour or pipette Figure 4 3 the appropriate amount see Table 4 2 of warm agarose lt 60 F onto the UVT gel tray that has been placed into the casting position in the casting chamber Immediately after pouring insert the desired comb or combs into the comb slots to form the sample wells Repeat with second gel tray if casting 2 gels Allow the gel s to solidify completely If a short running distance is required for proper sample separation then 3 combs may be used This expands the number of samples per run Thermo Scientific Section 5 Using the System Running Two Gels 1 Once the gels are completely solidified lift one tray out of the casting chamber Figure 5 1 and place it in the buffer chamber Figure 5 2 with the first comb closest to the cathode black side of the chamber The running position exposes the open ends of the gel tray and the agarose to the buffer Standard agarose should solidify completely in about 30 minutes If low melting point or a specialty agarose is used consult the instructions supplied with the product 2 To avoid damage to the sample wells gently rock the comb back and forth lightly to loosen then slowly pull the comb straight up out of the gel tray This rocking helps to avoid suction a
20. s the comb is removed Be sure to remove combs while immersed in buffer 3 Follow Step 1 and 2 above for the second gel tray Running One Gel 1 Once the gel is completely solidified lift the gel tray out of the buffer chamber from the casting position and turn 90 placing it back into the buffer chamber in the running position see page 4 Figure 5 amp 6 The running position exposes the open ends of the gel tray and the agarose to the buffer Standard agarose should solidify completely in about 30 minutes If low melting point or a specialty agarose is used consult the instructions supplied with the product Figure 5 1 Casting Position Figure 5 2 Running Position Thermo Scientific Horizontal Electrophoresis 5 1 Section 5 Using the System 5 2 Horizontal Electrophoresis Running One Gel continued 2 Carefully remove the comb or combs by tapping lightly to loosen Figure 5 3 and slowly lifting out away from the gel tray to avoid damage to the wells nn Figure 5 3 Carefully Remove Comb 3 Load the sample into the gel by using one of the following options Dry loading Loading the sample in gel without the presence of buffer a Remove the gel tray from the casting chamber b Load the sample into the gel but be careful not to puncture the bottom of the gel Place the gel tray into the buffer chamber in the running position see Figure 4 5 c Remove the second gel tray from the cas
21. the bottom tray Add buffer to the second fill line Continue to load samples Note To run one gel follow Steps a b cand d A Note It is recommended to always run a sample beren lane of a known standard ladder or marker to determine concentration and size of separated A16 fragments after the gel run and to aid in 6 557 photodocumentation and analysis Migration patterns and fragment sizes for commonly used DNA molecular weight markers are shown in aa Figure 5 4 A 2 322 2 027 Figure 5 4 Sample Lane Thermo Scientific Horizontal Electrophoresis 5 3 Section 5 Using the System Finishing Up 5 4 Horizontal Electrophoresis 4 Carefully slide the Supersafe lid with attached power supply leads onto the unit Figure 5 4 This will connect the power supply leads to the banana plugs to complete the circuit Plug the other end of the power supply leads into an appropriate power supply Turn on power supply See Recommended Running Conditions Table 3 1 When the gel run is complete and tracking dye has migrated as far through the gel as desired or to the end of the gel turn off the power supply and slide off the lid to disconnect the unit from the power source Carefully remove the tray s containing the gel wear gloves if ethidium bromide is present The UVT gel tray makes visualization and photography with a UV light source easy without the need to remove the gel from the tray When the gel ru
22. the unit with running buffer and attach to the power supply without a gel or gel tray in the unit The platinum wires on both sides of the unit should produce small bubbles as the current passes through If a complete circuit does not exist there will be little to no bubbles Contact Technical Services to schedule a repair Samples that appear to run backwards through the gel is caused by the tray being placed in the chamber in the reverse direction The tray should be placed in the chamber with the comb at the edge of the tray closest to the cathode side of the chamber Samples are not moving as expected through the gel remaining in the wells running backwards or diffusing into the gel Thermo Scientific Horizontal Electrophoresis 8 1 Section 8 Troubleshooting Problem Always make sure to allow the gel to solidify completely before moving the tray When the comb is removed from the gel the sample well unit or removing the comb To avoid damage to the sample wells gently rock the is ripped and damaged comb back and forth lightly to loosen then slowly pull the comb straight up out of the gel tray This rocking helps to avoid suction as the comb is removed The volume of running buffer used to submerge the gel should only be between 3 5mm over the gel surface Thw gel should be completely submerged to avoid the gel from drying out which can smear the bands and possibly melt the gel due to overheating If excessive running buff
23. ting chamber d Load the sample into the gel but be careful not to puncture the bottom of the gel Place the gel tray into the buffer chamber in the running position see Figure 4 5 e Carefully fill the buffer chamber with buffer to cover either one tray lower fill line or both gel trays up to the upper fill line Note To run one gel follow Steps a and b Fill the buffer chamber with buffer to cover one gel tray to the lower fill line A Thermo Scientific Section 5 Using the System Wet loading Loading the sample in gel when it is submerged in buffer a Remove one gel tray from the casting chamber b Place the gel tray into the buffer chamber in the running position c Pour running buffer into the unit to fill chamber and completely cover and submerge the gel Two Fill Lines are located on each unit to clearly mark the correct buffer level See Recommended Running Conditions Table 3 1 for approximate buffer volumes needed for your unit Too little buffer may cause the gel to dry out during the run while excess buffer may slow DNA migration in the gel d Load prepared samples into the wells Samples should be mixed with a sample loading buffer giving weight to the samples so that they drop evenly into the wells and contain tracking dye to monitor the gel run See Table 4 2 for approximate well volumes e Remove the second tray from the casting chamber and put into the buffer chamber directly on top of
24. ures associated with this symbol re gt Equipment being maintained or serviced must be turned off and locked off to prevent possible injury Hot surface s present which may cause burns to unprotected skin or to materials which may be damaged by elevated temperatures Marking of electrical and electronic equipment which applies to electrical and electronic equipment falling under the Directive 2002 96 EC WEEE and the equipment that has been put on the market after 13 August 2005 Lars This product is required to comply with the European Union s Waste Electrical amp Electronic Equipment WEEE Directive 2002 96 EC It is marked with the WEEE symbol Thermo Fisher Scientific has contracted with one or more recycling disposal companies in each EU Member State European Country and this product should be disposed of or recycled through them Further information on Thermo s compliance with this directive the recyclers in your country and information on Thermo products will be available at www thermofisher com Y Always use the proper protective equipment clothing gloves goggles etc VY Always dissipate extreme cold or heat and wear protective clothing Y Always follow good hygiene practices Y Each individual is responsible for his or her own safety Thermo Scientific Horizontal Electrophoresis iii Preface Do You Need Information or Assistance on Thermo Scientific Products If you do please contact us 8 00 a m t
25. ved baked or placed in a microwave To order RNase Away contact Molecular BioProducts 800 995 2787 U S and Canada or 858 453 7551 7000 250ml bottle 7002 475ml spray bottle 7003 1 liter bottle 7005 4 liter bottle Rnase AWAY is a registered trademark of Molecular BioProducts Horizontal Electrophoresis 6 1 Thermo Scientific Section 7 Reagents Information Selection of Reagents for Gel Electrophoresis 1 Agarose There are various types of agarose commercially available that may be used Besides standard ultra pure electrophoresis grade agarose there are also numerous low melting point products for easy sample recovery as well as specialty products formulated for specific uses i e to separate and or recover very small or very large fragments Table 7 1 Mobility range of DNA in different percentage agarose gels Agarose w v lt 0 1 Table 7 2 Amount of Agarose to prepare Gel volume is determined by the following formula and may be adjusted according to need or preference gel width cm X gel length cm X gel thickness cm ml of agarose Width of Gel cm Length of Gel cm Thickness of Gel cm Volume of Gel ml Horizontal Electrophoresis 7 1 Section 7 Reagents Information Table 7 3 Sample Volume D4 unit volumes of comb wells Thickness Width of e A Paro of Teeth Recommended loading volume ul fe Jon Pan slk pe fe je For different thicknesses of ge
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