B1A, B1, B2 and B3 Horizonal Electrophoresis Systems User Manual
Contents
1. p TWNOLLVNYSLNI ALNVSSVM SLINAOAd IMO 9131133195 S3HSI3 OINYSHL Thermo Scientific Horizontal Systems 9 2 Thermo Fisher Scientific 81 Wyman Street P 0 Box 9046 Waltham Massachusetts 02454 9046 United States www thermofisher com ThermoFisher SCIENTIFIC2. run Multiple sample loading configured for use with an 8 channel pipette is available by using the multi load tray see Section 8 To conserve agarose a wall comb may also be used to divide and use a smaller portion of the length of the gel tray If a wall comb is used pipette a bead of agarose along the bottom and side edges of the wall comb once it has been placed in the tray to seal the combs edges to the trays bottom and sides Once this bead is solidified the cooled gel may be poured as described Alternately regular tape cut slightly longer then the comb can be placed flat along the combs surface and the comb angled into place in the gel tray Extra tape is then placed on the outside of the comb in the excess tray area to reinforce the corners Allow the gel to solidify completely Thermo Scientific Thermo Scientific Section 2 Setting Up Migration Distance Run one sample set on a gel in each tray Run two sample sets on a gel of Run Length 1X equal length comb slots in each TAN irri tray YY JRun Length 2x Run three sample sets on a gel of equal length samples comb slots in each tray Run Length 3X Run Length 4X Run four sample sets on a gel of equal length samples comb slots Figure 2 3 Run Lengths in each tray and so on up to 12 rows Specific Tray Options for Each Model Model B1A Model B1 Two combs for two 4 9cm run lengths One comb for 12 9
3. to the anode end of the chamber This flow of bubbles displaces buffer 3 to create an effective recirculation within the chamber Unpack amp Check Your Order Before starting unpack the unit and inventory your order If any parts are missing contact Technical Services within 7 days of purchase Super Safe Lid Power Supply Leads UVT Gel Tray Buffer Chamber Gasket 2 1 0mm thick Double Sided ere ar Comb 2 1 5mm thick Figure 1 2 Exploded Parts Diagram B1A B1 amp B2 Parts List Buffer Chamber Combs 2 1 0 1 5mm thick double sided Super Safe Lid with attached Power Supply leads 2 Gasketed UV Transmissible UVT Gel Tray Specifications and Recommended Running Conditions Gel Size W x L cm 7x8 9x11 12x14 Buffer Capacity 400ML 600ML 800ML Time Requirements minutes 30 60 45 90 60 120 Voltage Requirements V 20 150 20 150 20 150 Thermo Scientific Thermo Scientific Section 2 Setting Up 1 Remove the SuperSafe lid from the buffer chamber The SuperSafe Lid is attached to the back of the unit at the junction of the lid s attached power supply leads to the banana plugs located on the unit To remove hold the front of the buffer chamber with one hand and pull the lid off sliding it off evenly by holding the center of the back of the lid 2 For shipping and convenient storage the gel trays are packaged inside the casting chamber To remove the gel
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5. 3 A DL are 4 to 0 2 DIN Sh Mosis da caca AGE ere ee 3 to 0 1 ci re net tus di ettet eo des 0 1 It should be noted an increased agarose gives better separation of small fragments and also bands very close together that tend to be more difficult to separate visualize and photograph A specialty agarose product formulated to increase resolution of low molecular mass samples may also be used Example A good mid range gel percentage would be 0 796 or 0 7g agarose in 100mls electrophoresis buffer TBE or TAE following heating and dissolving the agarose 10ul of ethidium bromide stock solution 5mg ml is added The gel would be run with compatible electrophoretic running buffer 1X TBE or 1X TAE that also contained ethidium bromide 1 liter of the running buffer would contain 100ul of this 5mg ml ethidium bromide stock solution Preparation amp 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 mM EDTA 50X stock solution pH 8 5 1X working solution 242g Tris base 40mM Tris acetate 57 1ml glacial acetic acid ImM EDTA 18 612 Na2EDTA 2820 MW 372 24 Distilled H20 to 1 liter final volume Thermo Scientific
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7. Model B LP ensures a flat casting and running surface The platform is 46cm x 36cm and is large enough to fit most applications One bubble level BBL 1 is included Replacement Parts Contact Technical Services to order replacement parts B1 Replacement Parts Item Description Catalog No Complete System BIA Complete System with Buffer Exchange Ports B1A BP AccessoHies oa A Catalog No Power Supply Leads PSL 5 Gasketed UVT Gel Tray BIA UVT Replacement Gaskets 1 pair External Caster trays not included B1A CST Leveling Platform 36cmW 46cmL B LP Bubble rel sausa erui Adr ca MeL cut BBL 1 8 2 Horizontal Systems Thermo Scientific Thermo Scientific B1 Replacement Parts Item Description Catalog No Complete System rallada Bl Complete System with Buffer Exchange Ports B1 BP Accessories Catalog No Power Supply Leido PSL 5 Gasketed UVT Gel Tray B1 UVT Replacement Gaskets 1 pair B1 GK External Caster trays not included B1 CST Leveling Platform 36cmW x 46cmL B LP Bubble dca ar BBL 1 B2 Replacement Parts Item Description Catalog No Complete System B2 Section 8 Section ti
8. and Check oue doa dure ee 1 2 Setting UP un eoe ar 2 1 Preparinesthe Gel u aa ur ae ra aa 2 2 Migration Distance aan debate ee as 2 3 Using the System 3 1 Specifications and Recommended Running Conditions 3 2 Finishing UP 4 1 Technical Tips cese eR a e 5 1 Troubleshooting rot r ee 6 1 Care and 7 1 Optional Equipment 0 0 e cece eee eee eee eee 8 1 Horizontal Systems V Thermo Scientific Section 1 General Information Introduction Horizontal Minigel Systems Models and B2 The Horizontal Minigel Systems are designed to provide flat even banding patterns and consistent results with hassle free gel casting The all in one design allows you to cast and run gels in the same chamber eliminating the need for additional casting equipment No tape grease agarose seals or other accessories are required All systems accommodate 2 comb positions allowing the user to run 2 series of samples equal distances simultaneously Convenient visualization strips have been added for easier sample loading and fluorescent ruled UVT gel tray helps in the precise photodocumentaion of each gel run Three gel sizes are available for increased sample capacity and running length Stand alone casting platforms are available for pouring up to 3 gels simultaneously while the chamber
9. comb ripped and damaged is removed Alternatively once casting is complete simply sub merging the gel with running buffer will help loosen the comb Using a higher percentage of agarose that forms a tighter gel matrix may remedy this problem as well The volume of running buffer used to submerge the gel should only be between 3 5mm over the gel surface Gel should be completely submerged to avoid the gel from drying out which The gel seems to run slower can smear the bands and possibly melt the gel s due to over under the usual running conditions heating If excessive running buffer 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 Thermo Scientific Horizontal Systems 6 1 Section 6 Troubleshooting 6 2 Horizontal Systems RN Check to see if the gasket is correctly seated in groove and even all the way around Remove gasket and reseat by smooth ing out gently with your thumb from one end to the other Gasket material may have a tendency to absorb salts from the running buffer After each use rinse the end gates under warm running water to bring back spongelike consistence of the gas ket material Gaskets may eventually become brittle with fre quent use Contact Technical Services to purchase replacement gaskets Agarose leaks into chamber when casting the gel Check to be sure that the unit is
10. never be autoclaved baked or placed in a microwave A Warning DEPC is suspected to be a carcinogen and should be handled with care To order RNase AWAY contact Technical Services Part Number 2 5 ssc ches ene Description TOOG a ae ee 250ml bottle 7002 a 475ml spray bottle 7003 Hase ande Barren 1 liter bottle PONTE RT 4 liter bottle Rnase AWAY is a registered trademark of Molecular BioProducts Horizontal Systems 7 1 Section 7 Care and Cleaning 7 2 Horizontal Systems Care Of Acrylic This list does not include all possible chemical incompatibilities and safe compounds Thermo acrylic products should be cleaned with warm water a mild detergent such as Alconox and can also be exposed to a mild bleach solution 10 1 In addition RNAse removal products are also safe for acrylic Contact Technical Services with any questions The following chemical compatibility chart is supplied for the convenience of our customers Although acrylic is compatible with most solvents and solutions found in the biochemical laboratory some solvents can cause substantial damage Keep this chart handy to avoid harm to your apparatus by the use of an inappropriate solvent Codes S Safe No effect except possibly some staining A Attacked slight attack by or absorption of the liquid slight crazing or swelling but acrylic has retained most of its strength U Unsatisfactory softened swollen
11. properly leveled for casting and running the gel by using the thumbscrews on the base Bands seem to be running at Thumbscrews should be adjusted until the bubble in the level an angle lines up with the levels center circle Always center the gel tray holder on the platform and cool the agarose to below 60 before pouring to avoid warping the UVT gel tray s Check that the platinum electrode wire is intact running flat and evenly across the outer corners and up the side to the junction of the banana plug area This problem could also be Samples seem to be running caused by regular casting with very hot agarose gel gt 60 unevenly in certain areas which may damage the gel tray over time Always cool the melted agarose to below 600 before casting to avoid warping the UVT gel tray s Warping the UVT 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 Samples do not band sharply used it may be necessary to completely solidify gels at and appear diffuse in the gel 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 Additional Sources For Reference Maniatis T E Fritsch and J Sambrook Molecul
12. trays hold the casting chamber firmly with one hand grasp the long sides of the UVT gel tray and pull up slowly at an angle with your other hand The trays fit snugly for leak proof gel casting therefore they may be tight Walking the tray upwards at an angle may be helpful The tightness will diminish the more the unit is used Additional Step for Model B3 Priming the Unit Fill the B3 chamber with enough buffer to fill both compartments and allow it to stand for about 15 minutes prior to running Fill the chamber at the cathode end black electrode first This will flush out trapped air in the hydrogen collector and recirculation tube Priming the unit is most important when using buffers of low ionic strength like TAE or NaPO4 This process minimizes the electrostatic repulsion between the hydrogen gas bubbles and the recirculation tube s surface Neglecting this step may result in decreased efficiency of the recirculation system Refer to normal Setting Up instructions for use following this priming step 3 To cast gels place the UVT gel tray into the buffer chamber in the casting position Figure 3 1 making sure the gel tray rests level and centered on the platform Be sure the gasketed ends of the gel tray press against the walls of the buffer chamber Thermo offers a leveling platform Catalog No B LP Section 8 if needed Figure 2 1 UVT Gel Tray Horizontal Systems 2 1 Section 2 Setting U
13. unaffected 4 There are four options that fit the use of a 9mm center to center pipette tip format The 9mm spacing represents _ SPT AN a 1x option micro well 1st Loading format By decreasing the 4 4 center to center distance factors of 9mm one can fit more samples in a given 2nd Loading _ 9 divided by 2 the 3x is 9 Micro Well Format 1X Micro Well Format 2X divided by 3 and the 4x is 9 divided by 4 Figure 5 3 Loading Options amount of space with the ability to use the same micro well format pipette The 2x is Therefore it is possible to have a greater number of teeth in a comb and maintain the use of the multichannel pipette by having the multichannel pipette fill every other well rather than every well This type of multichannel pipette format comb is called a 2x multichannel pipette format comb For example the 50 tooth comb for the A6 device has center to center distances between teeth of 4 5cm This means that a researcher would load lanes 1 3 5 7 9 11 13 and 15 with the first pass of the pipette and 2 4 6 8 10 12 14 and 16 with the second pass and so on until all of the lanes are filled Horizontal Systems 5 3 Section 5 Technical Tips 5 4 Horizontal Systems When using an 8 or 12 channel pipette the number of sample wells that can be filled must be a multiple of 8 or 12 A 25 we
14. Horizontal Systems Models B1A B1 B2 and B3 Operating and Maintenance Manual 7007309 Rev 0 Visit us online to register your warranty www thermoscientific com warranty 6 Owl Separation Systems Preface Model B3 MANUAL NUMBER 7007309 0 4 16 12 Transfer to Marietta was 2 2004 ccs REV ECR ECN DATE DESCRIPTION By Thermo Scientific Horizontal Systems 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 Caution All internal adjustments and maintenance must be performed by qualified service personnel Warning 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 shut down on disconnect circuit Do not move the unit unless the power source to the unit has been disconnected 4 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 Caution Running conditions for this unit should not exceed the name plate readings found on the lower buffer chamber 4 This Owl System is designed to meet IEC 1010 1 safety tandards IEC 1010 1 i
15. Section 5 Technical Tips TBE Tris borate with EDTA 89mM Tris base 89mM boric acid 2mM EDTA 10X stock solution 1X working solution 1088 Iris base ame 89mM Tris base 55g boric acid 89mM boric acid 7 44g Na2EDTA 2 20 MW 372 24 2mM EDTA or 40 ml 0 5 M EDTA pH 8 0 Distilled H2O to 1 liter final volume Do not adjust pH Buffer TAE Buffer Suggested Uses and Comments Use when DNA is to be recovered For electrophoresis of large gt 20kb DNA Applications requiring high resolution Has low ionic strength and low buffering capacity recirculation may be necessary for long runs gt 4hrs Buffer TBE Buffer Suggested Uses and Comments For electrophoresis of small lt 1kb DNA Better resolution of small lt 1kb DNA Decreased DNA mobility High ionic strength and high buffering capacity no recirculation needed for extended run times TBE buffer reacts with the agarose making smaller pores and a tighter matrix This reduces broadening of the DNA bands for sharper resolution Ethidium Bromide Ethidium bromide is ideal for the flurometric detection of nucleic acids in gel electrophoresis The addition of ethidium bromide to both the prepared gel and running buffer is a convenient way to monitor separation and keep a photographic log of gel runs Ethidium Bromide is prepared as 10mg ml in distilled water and used as a stock working solution of 5 0ug ml in the electroph
16. T gel trays and combs are sold separately Multi Load Tray Figure 8 2 Multiple Gel Caster Figure 8 1 Buffer Exchange Port Option for Models B1A B1 And B2 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 and will contain two separate port inserts packaged in a small plastic bag located inside the unit upon arrival Ports are attached to a user supplied pump amp Figure 8 3 Buffer Exchange Port Option Thermo Scientific Horizontal Systems 8 1 Section 8 Optional Equipment 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 you 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 Leveling Platform The three point leveling platform
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18. ar 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 BioTechniques Vol 10 No 2 1991 Boots S Gel Electrophoresis of DNA Analytical Chemistry Vol 61 No 8 April 15 1989 Thermo Scientific Thermo Scientific Section 7 Care and Cleaning Caution Organic solvents cause acrylic to craze or crack Clean all acrylic systems with warm water and a mild detergent do not use ethanol or other organic solvents to clean these products Do not autoclave bake or microwave your unit Temperatures over 50 C can damage the acrylic 4 Note If an RNase free electrophoresis system is desired there are various methods to rid the system of RNA contamination For fast and casy 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 diluted bleach This electrophoresis system should
19. cm 12x14 12x14 Buffer Capacity 400ml 600ml 800ml 1000ml Voltage Requirements V 20 150 20 150 20 150 20 150 Time Requirements minutes 30 60 45 90 60 120 60 120 Thermo Scientific Thermo Scientific Section 4 Finishing Up 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 SuperSafe lid to disconnect from the power source Carefully remove the tray containing the gel wear gloves if ethidium bromide is present The UV Transmissible UVT gel tray makes visualization and photography with a UV light source easy without the need to remove the gel from the tray 2 The buffer chamber SuperSafe lid UVT gel tray and combs should be rinsed under warm running water after each use An RNase DNase decontaminate may be used This electrophoresis system must never be autoclaved baked or microwaved Note Rather than staining with Ethidium Bromide other stains are now available which are less hazardous These include SYBR Green SYBR Gold and SYBR Safe While their excitation maxima are in the visible region there is a fair amount of excitation in the UV region so your current UV transilluminator will work with them Besides safety another advantage to using a light source with a frequency in the visible light region is that the transmission diffuser normally a quarts window on UV lights can be made of far le
20. cm run length Two combs for two 6 5cm run lengths Figure 2 4 Tray options Horizontal Systems 2 3 Thermo Scientific Section3 Using the System 1 Once the gel is completely solidified lift the tray out of the chamber turn it 90 in the running position see Figure 3 1 and replace it in the chamber with the first comb closest to the cathode side of the chamber This running position exposes the open ends of the agarose to the buffer Standard agarose should solidify completely in about 30 minutes If low melting point or a speciality agarose is used consult the instructions that came with the product 2 Pour enough compatible running buffer into the unit to fill the buffer chamber and completely cover and submerge the gel Correct buffer level is clearly marked on the units side wall as FILL LINE See Recommended Running Conditions 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 decrease DNA mobility and cause band distortion 3 Carefully remove the comb or combs by tapping lightly to loosen and slowly lifting straight up out of the gel tray to avoid damage to the wells 4 Loading the Sample in Gel Wet loading loading the sample in the gel when it is submerged in buffer a Place the gel tray into the buffer chamber in the running position b Pour running buffer into the unit to fill chamber and completely co
21. easy sample recovery as well as speciality products formulated for specific uses to separate recover very small or very large fragments etc To visualize and photograph the samples after the gel run for a permanent record 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 quickly and easily visualize and photograph the separated fragments following the gel run without the need for additonal staining If this is not added then following the gel run the gel may also be soaked in a concentrated ethidium bromide solution and rinsed for the same visualization The ethidium bromide is added to both the gel after heating and the electrophoresis buffer at a concentration of 0 5ug ml Warning Ethidium bromide is a potential carcinogen Care in handling the powder and stock solution must be taken Always wear gloves when handling the powder solutions and all gels that contain any amount of ethidium bromide Horizontal Systems 5 5 Section 5 Technical Tips 5 6 Horizontal Systems Mobility range of DNA in different percentage agarose gels Agarose w v Approximate range of separated DNA fragments kb ED A dra OR e eR rd cue 60 to 5 j5 MP 30 to 1 tr citt utet s Caras id docu mtr ar 12 to 0 8 ID etri eo bo OU EN Eat a 10 to 0 5 Ee 7 to 0
22. fic Section 8 Optional Equipment Comb Options Model B1 Recommended Loading Volumes Catalog Thickness Width of Gel Thickness Comb Type cf teeth lof Teeth Teeth ss ee Ju 78 5 90 6 525 32 750 46 Loading volume is calculated as 75 of total well volume see page 15 Comb Options Model B2 amp B3 Recommended Loading Volumes epe ESL 283 fe fa ae pa qe es NN epe qur B2 Prep 106 5 120 55 415 20 710 34 1000 48 Loading volume is calculated as 75 of total well volume see page 15 8 amp 12 Channel Pipette Format Horizontal Systems 8 5 Section 9 Warranty Information L006 OSI 2176 0 uoneuuojul JO Jojnqujsip Jno JOeNUOD YSN eui episino eio eds pue sonas uonejado AjueJeM Juswd nba uo suonsenb noA Jamsue peaJ 9J 9M 9 7 E2E 0PZ 1 Jo epeue pue VSN 158y 8 y 008 1 1e jueuniedeq samas peo aseajd s eor ues jueuudinbe y a9ueua julew anuanald pue jueuudinboe eap Ajjnjaseo paud jueuudinboe SAISUBYSJGWOD uj djay peaiJ si sejes INOA SjonpoJd Jo sso JO S JOJd 150
23. is in use Owl offers a wide variety of combs including options that can double your sample capacity by doubling the number of sample wells preparative combs and wall combs to run gels in smaller sizes Custom combs are also available Self Recirculation System Model B3 This system has a unique built in recirculation system that offers convenience and versatility The recirculation system prevents formation of pH and ionic gradients for high resolution and uniform reproducible results Ideal for long runs multiple sample sets or RNA gels this system delivers clear results for samples run over long time periods It also eliminates uneven migration band distortion or disassociation of pH dependent glyoxylated RNA molecules that can result when ionic depletion occurs 1 52 RAS e E m Figure 1 1 Self Circulation System Horizontal Systems 1 1 Section 1 General Information 1 2 Horizontal Systems Self Recirculation System Model B3 continued The self recirculating system reduces formation of pH gradients so you get high resolution and uniform reproducible results The self contained recirculation system requires no external pumps tubing or stir bars As shown in the diagram a trap at the cathode end of the buffer chamber 1 collects the hydrogen bubbles produced at the electrode during electrophoresis The bubbles are then shunted into a conduit tube 2
24. le volume for a horizontal gel 1 Gel volume which is Width x Length x Gel Height and uses centimeters and 2 Sample volume which is Tooth Width x Comb Thickness x Apparent Well Height and uses millimeters Thermo Scientific Thermo Scientific Section 5 Technical Tips How to Determine Well Sample Volume continued Gel height is generally set to a height between 0 25 cm and 1 0 cm Therefore once you choose the height the volume is the gel dimensions given in the catalog for each gel box I D times this height Once the gel height Hg is chosen the well volume and then the sample volume can be calculated The well height Hw is 1 5 mm less then the A gel height Hw Gel Height 1 5mm Using the well height the GEL TRAY BOTTOM volume of the well is calculated Vw _ Well Height Tooth width x Figure 5 5 Determine Volume comb thickness The loading volume is a 0 75 safety factor applied to the well volume Vs Vw 75 GEL TRAY SIDE Hg GEL HEIGHT SPACE For Owl combs there are two thicknesses 1 0mm and 1 5mm This is the depth The width of the well is determined by the number of teeth For a given gel box as the number of teeth increase the volume of each tooth decreases Reagent Information There are various types of agarose commerically available that may be used Besides standard ultra pure electrophoresis grade agarose there are also numerous low melting point products for
25. ll micro well format comb would have one extra sample and a 50 well micro well format comb would have 2 extra samples which a researcher could fill with a single channel pipette and is generally used for standards Why Recirculate Buffer During electrophoresis gradual ionic depletion of the running buffer forms an ionic and pH gradient across the system acetate and phosphate buffers are especially prone to ionic depletion Such gradients can cause uneven migration and banding patterns or cause pH dependent glyoxylated RNA molecules to disassociate Buffer recirculation ensures uniform ionic strength throughout the system Comparison of buffer pH with and without recirculation during agarose gel electrophoresis 14 12 50ng samples of Hind 5 i III digested DNA were E 8 run on duplicate gels 6 with and without buffer 3 recirculation pH 0 measurements were 0 50 100 150 200 250 taken at the anode and Time min e electrode hode ends at vari Ole recirculated catnode ends at various AT electrode recirculate Figure 5 4 Comparison O electrode recirculated time intervals and plotted against time Running condition 1 agarose gel in 10mM NaH2P04 pH 7 0 114V constant voltage Sample Well Comb Configuration Hg height of gel used Hs height of well used for sample volume Hw well height How to determine well sample volume There are two volumes to consider when determining the samp
26. nt 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 unit 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 Systems Thermo Scientific Thermo Scientific Section 1 Section 2 Section 3 Section 4 Section 5 Section 6 Section 7 Section 8 Table of Contents General Information 1 1 a a eA A a ER ANE a 1 1 Unpack
27. ore heating In general add powdered agarose to distilled water and swirl to mix Make sure all the powder is equally wet to ensure proper melting Heat in a microwave oven boiling water bath or hot plate with occasional swirling to melt and mix completely Cool agarose liquid to below 60 and cast Note Gel should be cast no thicker than 5mm to avoid fuzzy banding High percentage gels may thicken and solidify rapidly and should be cast while still a liquid Bands are not sharp clear and even Check that a complete power circuit is achieved between the unit and the power supply Platinum wire and banana plugs Samples are not moving as should be intact To test simply fill the unit with running buffer expected through the gel remain and attach to the power supply without a gel or gel tray in the ing in the wells or diffusing into Junit The platinum wires on both sides of the unit should pro the gel duce small bubbles as the current passes through If a complete circuit does not exist there will be few to no bubbles Contact Technical Services to schedule a repair Always make sure to allow the gel to solidify completely before moving the gel tray unit or removing the comb To avoid damage to the sample wells gently rock the comb back and When the comb is removed from forth lightly to loosen then slowly pull the comb straight up out the gel some sample wells are of the gel tray This rocking helps to avoid suction as the
28. ore information Wall Comb The wall comb is used in your existing U V Transmissible UVT gel tray to allow the ability to cast smaller gels using the existing gel tray and the comb slots There are various ways to use the wall comb to ensure a leak proof seal These two are the fastest and easiest Thermo Scientific Thermo Scientific Section 5 Technical Tips Comb Options continued Tape Method Using casting tape transparent tape or masking tape cut a piece long enough to cover the full length of the wall comb with about 1 2 overhang at each end Half the width of the tape should be free Firmly press the tape all along the comb leaving the three open edges loose Place the taped comb into the gel tray at the desired comb slot position The taped side should be facing away from where the gel will be cast While placing the comb angle it so the loose taped edge is free Once positioned into the gel tray firmly press the tape to the bottom and sides of the gel tray to form a leakproof seal Small pieces of tape may be added to the corners afterwards to reinforce the edges Add cooled lt 60 slightly thickened agarose to the gel tray and allow to solidify completely To remove comb gently remove excess tape and loosen tape from the bottom and sides of the gel tray Carefully pull comb straight up and out of the comb slot Note The edge of the gel may appear irregular once submerged in running buffer the gel run will be
29. oresis buffer and gel Mix ethidium bromide powder or tablet thoroughly into solution checking for any precipitate and store at room temperature protected from light Thermo Scientific Horizontal Systems 5 7 Section 5 Technical Tips 5 8 Horizontal Systems Amount of Agarose to Prepare Gel volume is determined by the following formula and may be adjusted according to need or preference Amount of Agarose gel width cm x gel length cm x gel thickness cm ml of agarose Agarose Volume in ml per gel thickness in cm Agarose Gel Loading Buffer Samples are prepared and combined with gel loading buffer before being applied to the prepared gel Sample buffer usually contains similar components to the running buffer dyes for visibility and glycerol to provide some weight to the samples This increased sample density and color allows easy visualization of the samples and 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 The most commonly used loading buffer is glycerol bromophenol blue and xylene cyanol Thermo Scientific Section 6 Troubleshooting m qm Always follow the proper procedure for preparing the agarose product according to the manufacturers instructions When preparing the agarose be sure all the agarose powder is in solution bef
30. p 2 2 Horizontal Systems 4 Preparing the Gel Using electrophoresis grade agarose and compatible electrophoresis buffer the gel may be prepared in various ways The percentage of agarose and the electrophoretic buffer used is determined by the size of the samples to be separated and further recovery of the samples see Table 5 1 page 16 The agarose and buffer are mixed and heated Figure 2 2 Pouring Gel over a heat source in a microwave oven or in an autoclave until the agarose is completely dissolved The prepared gel then must be cooled to below 60 C before casting to avoid warping the UVT gel tray due to excessive heat If numerous gels are to be run in one day a large volume of gel may be prepared and placed in a covered bottle stored between 40 60 C in a water bath This provides a ready gel supply in a warm liquid form that will solidify quickly when gels are cast For further tips on sample preparation and visualization see Section 5 Pour or pipette the correct amount see page 5 8 of warm agarose lt 60 C onto the UVT gel tray that has been placed in the casting position in the buffer chamber Immediately after pouring insert the desired comb or combs into the comb slots to form the sample wells If only a small portion of gel is required for proper sample separation multiple combs may be used to run 2 sets of equal distance samples simultaneously expanding the number of samples per gel that may be
31. rd the buffer chambers with the gasketed end gates removed and slowly fill the chamber with buffer Thermo Scientific Horizontal Systems 5 1 Section 5 Technical Tips 5 2 Horizontal Systems Comb Options Standard 1 0mm and 1 5mm thickness for all models Combs are hand fabricated for high quality precision in low volumes Each comb has an acrylic spine with Lexan teeth Double Sided im Standard Double sided molded combs combine 1 0mm and 1 5mm tooth thickness on one comb These combs provide greater precision due to the exact manufacturing technique which provides greater control over tooth size and spacing than the traditional machining Double Sided methods The number of teeth and arrows that point to the designated thickness is molded onto the spine for easy identification A raised section of the spine helps the user grip the comb when removing it from the gel Thermo s combs are molded from durable polycarbonate that holds up through years of use Preparative Preparative Figure 5 2 Combs Preparative combs are manufactured with an acrylic spine and Lexan teeth Used for extremely large samples Multi Load Comb For use with 8 12 channel pipettes These unique combs are designed to allow accurate easy loading from a 96 well plate Custom Combs Call Technical Services for m
32. s an internationally accepted electrical safety standard for laboratory instruments Material in this manual is for information purposes only The contents and the product it describes are subject to change without notice Thermo 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 Scientific All rights reserved i Horizontal Systems 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 procedures 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 ERA This product is required to comply with the European Union s Waste Electrical 82 Electronic Equipment WEEE Directive 2002 96 EC It is marked with the WEEE symbol Thermo Scientific has contracted with one or more recycling dispo
33. sal companies in each EU Member State European Country and this product should be disposed of or recycled through them Further information on Thermo compliance with this directive the recyclers in your country and information on Thermo Scientific products will be available at www thermo com Y Always use the proper protective equipment clothing gloves goggles etc Y 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 Systems iii Preface Do You Need Information or Assistance on Thermo Scientific Products If you do please contact us 8 00 a m to 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 equipme
34. slowly dissolved D Dissolved in seven days or less Thermo Scientific Section 7 Care and Cleaning Table 7 1 Chemical Compatibility for Acrylic Based Products PAS 285 Bee _____ ESC em _____ EEC fu je poenae eese p ja e o CT E pnm Ju hmm E ESTE E pem 5 _ DN emen ____ ee _ CEIC E s semen _ NEO CN n ean eem 5 _ names qr Hydrogen peroxide 28 solution e Te tnus s _ msn s quee ESO CU CN _ Denn DI uem p EEN _ umma frer O Deum E quen CO ma I This list does not include all possible chemical incompatibilities and safe compounds Acrylic products should be cleaned with warm water a mild detergent such as Alconox and can also be exposed to a mild bleach solution 10 1 In addition RNAse removal products are also safe for acrylic Contact Technical Services with any questions Thermo Scientific Horizontal Systems 7 3 Section 8 Optional Equipment Multi Load Tray amp Combs Multiple sample loading configured for use with an 8 channel pipette is available by using the multi load tray B2 RL and combs B2 RL 9D Multiple Gel Caster Pour multiple gels while the buffer chamber is in use UVT gel trays fit snugly between the walls of the heavy duty gel caster BLA CST B1 CST amp B2 CST Additional EasyCast UV
35. ss expensive materials and therefore made much larger Clare Chemical Research http www clarechemical com transilluminator htm makes several of these blue light transilluminator under the name of Dark Reader Dark Reader Model Viewing Surface Compatible Owl Units DR45M 14 x 21 All but A6 A3 1and D3 DR88M 21 x2b cm All but A6 and A3 1 Horizontal Systems 4 1 Section5 Technical Tips Running A Standard Ladder It is recommended to always run a sample lane of a known standard ladder or marker to determine 23 130 concentration and size of separated fragments after the gel run and to aid in photodocumentation and 9 416 analysis 6 557 Migration patterns and fragment sizes for commonly used DNA molecular weight markers are shown in this figure 4 361 Loading Samples It is sometimes easier to load the sample wells dry before placing buffer into the buffer chamber After 2 322 the gel solidifies if cast within the buffer chamber 2 027 remove the gel tray from the buffer chamber and place the tray on the lab bench Carefully remove the sample combs by tapping and lifting straight up Samples mixed with loading buffer that does not contain dye may be easier to load dry especially in larger gel units to Figure 5 1 Ladder avoid cross contamination After loading all sample lanes place the gel tray into the buffer chamber in the running position with the gel edges facing out towa
36. tle Complete System With Buffer Exchange Ports B2 BP Accessories sack sts he Catalog No Power Supply Leads PSL 5 Gasketed UVT Gel Tray B2 UVT Gasketed Multi Load UVT Gel Tray Slate aia B2 RI UVT Gasketed Multi Load UVT Gel Tray 12 Slots With 12 Combs B2 RI 9D B2 RL Replacement Gaskets 1 pair B2 GK External Caster trays not included B2 CST Leveling Platform 36cmW x 46cmL B LP B bbleleyel ous t tee ee ae BBL 1 Horizontal Systems 8 3 Section 8 Optional Equipment 8 4 B3 Replacement Parts Item Description Catalog No Complete System B3 Accessories Catalog No Power Supply Leads PSL 5 Gasketed UVT Gel Tray B2 UVT Gasketed Multi Load UVT Gel Tray I2 Slots iii be B2 RL UVT Gasketed Multi Load UVT Gel Tray 12 Slots With 12 Combs B2 RL 9D B2 RL Replacement Gaskets 1 Pair B2 GK External Multiple Casting Chamber trays not included Leveling Platform 36cmW x 46cmL B LP Bubble Level acer BBL 1 Comb Options Model B1A Horizontal Systems B2 CST Recommended Loading Volumes Width of Gel Thickness Teeth 17 26 34 51 ae pes fan far ae ws fe bs ma qne Loading Volume is calculated as 75 of total well volume see page 15 Thermo Scientific Thermo Scienti
37. ver and submerge the gel c 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 Note 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 containing tracking dyes to monitor the gel run See available comb section for approximate well volumes Comb Options Section 5 Horizontal Systems 3 1 Section 3 Using the System 3 2 Horizontal Systems Note It is wise to always run a sample lane of a known standard ladder to determine concentration and size of separated fragments after the gel run and to aid in photo documentation and analysis see Section 5 A Casting Position Running Position Gasket Figure 3 1 Tray Positions 5 Carefully slide the SuperSafe lid with attached power supply leads onto the unit This will connect the power cords to the banana plug electrodes Plug the other end of the power supply leads into an appropriate power supply completing the circuit The gel is now a resister in the circuit 6 Turn on power supply Refer to table below for running conditions Carefully monitor the gel run to avoid samples running into the path of another set of samples Specifications and Recommended Running Conditions Gel Size W x L in
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