S2 Sequencing Gel Electrophoresis Apparatus user manual
Contents
1. Casting Clamp 3 1 3 Pouring the Gel Using Gel Sealing Tape 1 Prepare the acrylamide gel solution see table 1 for the approximate volumes required for different gel thicknesses Note Most gel formulations allow approximately 10 min before polymerization Polymerization time can be extended by reducing the amount of TEMED added Warning Acrylamide is a known neurotoxin Consult the Material Safety Data Sheet for more information Table 1 Approximate Gel Solution Volume Requirement for Different Gel Thicknesses Gel thickness mm Volume required ml 0 2 35 0 4 65 0 8 130 1 6 260 awedge 140 a0 4 to 1 2 mm thick Operating Instructions 2 Use a large syringe squirt bottle funnel or beaker to pour the gel solution between the glass plates Hold the assembled plate sandwich at a 45 angle on one bottom corner so that the gel solution flows evenly down along the lower side spacer Maintain a constant even flow to reduce the chance of forming bubbles in the solution As the gel sandwich fills gradually lower the glass plates until they rest on the bottom edge approximately at a 5 angle from the bench The gel mold should be slightly overfilled to ensure complete polymerization at the top Note If a bubble forms while the gel is being poured raise the glass plates into a vertical position and either tip the gel solution away from the bubble or carefully tap the plate sandwich at the bubble to mak2. Progue ts viii n ada Gein eale 14 7 Additional Information ccc ceceeeeeeeeeeeeeeeteeeeeeeeeeeeteees 15 7 1 Care and Handling eeceeecceeseeeeeeeeneeeeeeeeeeceeeeeaeesaeeeeaeeseeeeeeeenaeeeeeeaas 15 7 2 Maintenance scissor saute oat 15 7 3 Specifications oo ee eeceeeeeeeeeeneeeeeeteeeeeeeeeaeeseeeeaeesseeesaeeseeeeeneeenaeeeeetias 16 7 4 Warranty 7 5 Declaration of Conformity and CE Mark ccceeseeseeeeeeeeeeetenreeeeeeneees 16 Figures 1 Model S2 Sequencing Gel Electrophoresis Apparatus cceeeeeeeeereeee 2 2 Taping Glass Plates ecceeceseseseeeeeeeeeneeceaeeeeeeseaeeseeeeaeeseeseaeeeieeeeeeeeeeeeaees 3 3 Assembly Using the S2 Casting Clamp ceeceeseeeeeeeereeteeeeeeeneeeeeeeeeeenaeens 4 4 Gel Casting Configuration of Sharkstooth COMD cccecceeeeeeteeeteeeeeenees 5 5 Sample Loading Configuration of Sharkstooth COMD ccccsseeseeeeeeees 6 6 Sharkstooth Comb Sample Loading Technique ccceeceeseeeeeeeteeeereeeees 8 7 Upper Platinum Wire with Carrier cccceeceeseeeseeeeeeeseeeeeceeeeeeeeneeteneeeeetens 15 Table of Contents Tables 1 Approximate Gel Solution Volume Requirement for Different Gel Thickn SSes cccccsccceseeceeeeeeceeeeeeeseneeeeseeeesceeeessaeeessneeetseees 4 2 Recommended Electrophoresis Power Settings for Different Gel Thickn SSes ccccccceseeeceeeeeeeeseeeeeeeeeeessaeeescaeeeessaeeesseeeeeteees 8 3 Samp
3. casting clamp does not require the use of accessory spring clips along the sides of the gel sandwich Leave the glass plates in their near horizontal position until the acrylamide has polymerized Remove the top clamp if used and carefully slide the comb from between the glass plates Operating Instructions 7 Remove the glass plates from the casting clamp Pull the casting clamp off the glass plates starting at the top and working your way down Take care in that there may be some unpolymerized acrylamide in the bottom of the casting clamp Rinse the top of the gel with electrophoresis buffer to remove any unpolymerized acrylamide Rinse the comb with deionized water If you are using a sharkstooth comb reinsert the comb between the glass plates with the teeth toward the gel Insert the comb until the teeth just make contact with the surface of the gel figure 5 Do not allow the teeth to pierce the gel A very slight indentation of the gel should be visible when the comb is properly inserted Do not slide the comb laterally after it has come in contact with the gel To fill the gel from the bottom assemble the gel plates so that when the casting clamp is laying down on the molded feet the short glass plate is on top 1 4 Place the assembled gel sandwich on a level surface resting on the molded feet of the casting clamp Open the bottom fill port and insert a Leur fitting Attach a syringe of suitable reserv
4. gel Operating Instructions 3 1 2 Preparation of Glass Plates Using the S2 Casting Clamp The S2 Casting Clamp can be used in place of the gel sealing tape and spring clips to seal the edges of the glass plates while casting the gel Assemble the glass plates as before and place the assembled sandwich in the casting clamp following the instructions below i Make sure that the inner surface of the casting clamp is clean of all dried acrylamide and grease Hold the casting clamp vertically with your thumbs resting at the top of the molded handles and your index fingers over the molded feet flex the clamp outward in a bow like manner so that the bottom of the clamp can fit onto the bottom of the gel sandwich Fit the gel sandwich snugly into the bottom of the casting clamp Start at one corner and seal towards the other corner After the gel sandwich is seated into the bottom of the clamp fit the sides of the clamp onto the sides of the gel sandwich Start at the bottom of one side using your hands to smooth the clamp onto the gel sandwich Repeat this for the other side of the gel sandwich When properly placed the sides of the casting clamp will extend to within 4 5 mm of the top of the short glass plate See figure 3 If the sides of the clamp should extend further than this reposition the sides of the casting clamp so that they are at the top of the short glass plate Glass plates Figure 3 Assembly Using the S2
5. supply do not exhibit reasonable friction or can be rocked easily replace the plugs or power cords e f power cords show any signs of wear or damage e g cracks nicks abrasions or melted insulation replace them immediately e Examine the electrode mounting nuts to ensure that they are tight and free of corrosion If mounting nuts are corroded they should be replaced 2 Gaskets e Regularly inspect the silicone gasket for cuts or tears and replace as needed 3 Aluminum plates e After each use rinse the aluminum plates thoroughly with deionized water to remove salts and urea Failure to keep plates properly clean may affect performance and introduce a potential electrical hazard 15 16 Additional Information 7 3 Specifications Installation Category I W GIGI sce deecsvadecoeiat cxusetagesseteas Seen Se ciateee a chee montane ented 6 32 kg 13 9 Ib Apparatus Dimensions W x D x H scceseseeeeeseeeeeeeeeeenees 42 2 x 21 6 x 44 5 cm 16 6 x 8 5 x 17 5 in Gel dimensions W x A ensisi ia 31 0 x 38 5 cm 12 2 x 15 2 in COmMStructiOn ccscceeeeeeeeeseeeees Flame retardant ABS acrylic aluminum silicone Electrode mate nda cis ssni iaire a oE Eaa iaa TR E platinum Maximum gel thickneSs eescceceseeeeenereeeeneeeeseeeeseneeeeeneeeenaeeeseeeeeeneeereneees 1 6 mm Working buffer volume ccceecceesenceeeeeeeeeeeeeeeeeneeeeeeeerseneneneaeesneeeseseaeesnene
6. 5 068 21035 076 21035 084 21035 092 21035 100 21035 118 21035 126 Please call the TECH LINE 21105 317 21105 341 21135 134 21045 018 21046 016 21045 026 11034 014 21105 432 21105 473 11032 018 11032 026 11098 019 21047 014 13 Related Product Cat No Products Replacement Parts Power Cords 48 in pair except Europe 11099 041 Power Cords 48 in pair Europe only 11099 025 Silicone Gasket 21960 018 Lower Buffer Chamber Banana Plug Replacement Kit 21105 101 Upper Buffer Chamber Banana Plug Replacement Kit see figure 7 21105 127 Includes cap nut O ring and Banana Plug Upper Platinum Wire with Carrier 21113 014 Lower Platinum Wire with Carrier 11114 014 Upper Buffer Chamber Lid 31115 017 Lower Buffer Chamber Lid 11116 019 Gel Clamp Replacement Kit 11958 352 Drain Hose Replacement 21121 017 Drain Knob Replacement Kit 21120 019 Includes knob 4 flat washers Reagents GEL MIx 6 6 Sequencing Gel System 15543 010 GeEL Mix 8 8 Sequencing Gel System 15545 015 GeL MIx RUNNING MaTeE 10X TBE Buffer 15546 013 Additional power supplies prepared acrylamide solutions buffers and other reagents for use with the Model S2 Apparatus are available Please consult the Life Technologies Catalogue and Reference Guide for additional information Platinum wire O ring Wire carrier Banana plug Cap nut Gel clamp Figure 7 Upper Platinum Wire with Carrier Addi
7. GIBCOBRL Instruction Manual Model S2 Sequencing Gel Electrophoresis Apparatus CAT SERIES 21105 Lire TECHYOLOGIES Essential Technologies for the Science of Life Table of Contents 1 Notices to CUSTOMER ccc cc ec eseeeeeeeeeeeeeeeeeeeeneeteneeeenseneeeees 1 1 1 Important Information eee eeeeeeeeeeeeenneeeeeeeeteeeeeeaeeeseaeeeseneeseeneeeeee 1 L2 Warnings aap SH le le adapted Sede asd ieee ede deed 1 2 Overview jcc boo oo eee A ee 2 2 1 Description sireisas a iaa e E aaaea A pa iaaea Sii 2 2 2 OMPONEN S iinne ereas a aenn aA NE E EEEE a EE Raa 2 3 Operating INStructions 3 2 c ce5cce eee eda 3 3 1 Gel Casting eee ET 3 3 1 1 Preparation of Glass Plates Using Gel Sealing Tape 3 3 1 2 Preparation of Glass Plates Using the S2 Casting Clamp 4 3 1 3 Pouring the Gel Using Gel Sealing Tape ceeceeeeeeseeeteeeees 4 3 1 4 Pouring the Gel Using the S2 Casting Clamp ccceeeeeeee 6 3 2 Electrophoresis issesisceenchs new nike ieee Slee 7 3 2 1 Pre Electrophoresis ec eesceeeseeeeeneeeeeneeeeesneeeesaeeeseneeeensaeeeeaes 7 3 2 2 Loading Samples s ccescsecsehete At aesnediseven i eee ieee 8 32 9 Electrophoresis nesca e a E Eai 8 3 2 4 Post Electrophoresis cscccesecceeeseeeeeseeeeeeeneeseeeeeeeeeeeseneneeeeeees 9 4 Troubleshooting Guide cece cece ects eeeeeeeeeeeeeeeeeeeeneees 10 5 References 2 523 Senden eee 13 6 Related
8. ay then be discarded separately After disposing of all buffer rinse both chambers of the tray thoroughly with deionized water and place the tray back in the sequencing unit Disassemble the gel sandwich by prying off the short or siliconized glass plate working gently from a bottom corner with a thin spatula For autoradiography transfer the gel to a solid backing such as paper or used film Rinse combs side spacers and glass plates to remove buffer and gel fragments and dry all components before storing 10 Troubleshooting Guide Many equipment problems can be solved by carefully following the instructions in this manual Some suggestions for troubleshooting are given below Should these suggestions not resolve the problem please call the Life Technologies TECH LINE numbers listed on the back cover of this manual If the unit must be returned for repair contact the Customer Service Department or your local distributor for shipping instructions Please include a full description of the problem Problem Gel Solution leaks from the bottom The glass plates crack The upper buffer chamber will not drain The gel dye front is not straight Buffer leaks from the upper buffer chamber Sparks or burn marks appear at edges of aluminum plate Suggested Solution Lower the angle of the glass plates when pouring the gelduring casting Check that the inner surface of the casting clamp is clean and free of debris and
9. cers Do a double loading Use ammonium persulfate powder to make solution Alternatively remove persulfate solution the gelatin capsule from the ammonium persulfate capsule before dissolving 12 Allow the gel to polymerize at a 5 angle instead of flat Use an electrophoresis unit with an aluminum plate to disperse heat evenly Apply the pressure points of the binder clips only over the spacer Polymerize the gel at an angle of lt 10 Do not over tighten clamps on the apparatus Make sure that spacers are pushed against the polymerized acrylamide Do not use bottom clamps without a bottom spacer Do not pull plates together while taping the gel Make sure glass plates are seated flat in the electrophoresis unit Make sure the gel electrophoresis unit is level 11 12 References Anderson S 1981 Nucl Acids Res 9 3015 Bankier A T Weston K M and Barrell B G 1987 Methods Enzymol 155 51 Life Technologies Inc 1985 M13 Cloning Dideoxy Sequencing Instruction Manual Blakesley R 1983 Focus 5 1 1 Johnson Dow L Mardis E Heiner C and Roe B A 1987 BioTechniques 5 754 Kuebbing D 1983 Focus 5 2 1 Martin R 1987 Focus 9 1 8 Maxam A M and Gilbert W 1980 Methods Enzymol 65 499 Sanger F Nicklen S and Coulson A R 1977 Proc Nat Acad Sci USA 74 5643 Smith A J H 1980 Methods Enzymol 65 560 Tullius T C Dombroski B A C
10. e strick liability breach of warranty or any other theory arising out of the use of the product listed herein Life Technologies reserves the right to make improvements in design construction and appearance without notice 7 5 Declaration of Conformity and CE Mark Note The information outlined in this section applies only to customers located in the European Union EU The EU is currently comprised of 15 member countries This laboratory apparatus is identified with the CE mark This mark indicates that the product complies to the following EU Directives and Standards Application of Council Directive s 73 23 EEC Low Voltage Directive Standards EN 61010 1 1993 Product Safety EU Representative Life Technologies Ltd EU Address 3 Fountain Dr Inchinnan Business Park Paisley PA49RF Scotland A copy of the Declaration of Conformity certificate is available upon request PartNo 10451 Lot No 1088518
11. e a large syringe squirt bottle funnel or beaker to pour the gel solution between the plate sandwich Hold the assembled plate sandwich at a 25 35 angle on one bottom corner so that the gel solution flows evenly down along the lower side spacer Maintain a constant even flow to reduce the chance of forming bubbles in the solution As the gel sandwich fills gradually lower the casting clamp until it rests on the molded feet approximately a 5 angle The gel mold should be slightly overfilled to ensure complete polymerization at the top Before the acrylamide polymerizes insert a well forming comb into the top of the gel If you are using a sharkstooth comb insert the flat edge of the comb between the glass plates to a depth of 2 to 3 mm below the top of the short glass plate figure 4 Keep the flat edge shallow to simplify subsequent reinsertion of the comb in the sample loading teeth down configuration and to allow loading with a micropipet Mark one end of the comb so that you can keep it in the same left to right orientation when loading samples later If you are using a DELRIN square toothed comb available separately insert the teeth 4 to 5 mm below the edge of the short plate For improved comb sealing clamp the glass plate sandwich over the comb with a top clamp over the center of the comb It is important that the top clamp be placed over the comb only Clamping unsupported glass will distort the thickness of the gel The
12. e it rise to the surface Once all bubbles have been removed return the glass plates to their previous position Before the acrylamide polymerizes insert a well forming comb into the top of the gel If you are using a sharkstooth comb insert the flat edge of the comb between the glass plates to a depth of 2 to 3 mm below the top of the short glass plate figure 4 Keep the flat edge shallow to simplify subsequent reinsertion of the comb in the sample loading teeth down configuration and to allow loading with a micropipet Mark one end of the comb so that you can keep it in the same left to right orientation when loading samples later If you are using a DELRIN square toothed comb available separately insert the teeth 4 to 5 mm below the edge of the short plate SLASSSLSLSS N N N 4 7 Figure 4 Gel Casting Configuration of Sharkstooth Comb A top clamp and spring clips are recommended for securing the comb plate sandwich Place two spring clips over the edge of the plates to clamp on the comb figure 4 Do not place the pressure points of the spring clip over the side spacer Place the top clamp over the top of the glass plates and clamp the center of the top edges of the plate securely against the comb Proper placement of the spring clips and top clamp will force the plates against the comb ensuring a tight fit of the comb following polymerization If you use sprin
13. ee preceding comments The following information addresses some of the causes of the common problems for sequencing gel electrophoresis Problem Blurry bands Smeared bands Wavy bands Glass plates crack during electrophoresis Lack of resolution in the upper half of the gel Smiling gels Corresponding bands in outer lanes electrophor ese slower than inner lanes Frowning gels Corresponding bands in outer lanes electrophor ese faster than inner lanes Lanes curve outward toward bottom of the gel Bands are not even from lane to lane Possible Cause Urea diffusing into the wells Formamide in sample buffer degraded Excessive salt in the sample Radiolabel properties lead to scattering of signal Gel was overloaded Urea diffused out of gel Sample electrophoresing with the ion in front Gel temperature too high Sample electrophoresing at the surface of Formamide in sample buffer degraded Excessive salt in the sample The surface of the gel did not polymerize evenly Sharkstooth combs punctured the surface of the gel Gel is too hot Limitations in the resolving capabilities of acrylamide Capsules used to make 10 ammonium Gel thickness is not uniform Gel temperature is not uniform Improper clamping during casting of the gel Improper clamping of the gel into the electrophoresis chamber Improper gel casting Gel sandwich is not level Suggested Solution After pre electrop
14. eesees 1L Comb included 14 cm sharkstooth 25 pt 0 4 mm thick Voltage Range icicsscctsceeoecesdecctesl adeitecasasscpuneecbixccie tees AEE Ea 2000 VDC Max Current R amp Ange ccccccccseceesseceesseeeeseeeeeseneeesseeeesssaeeesnseeeseeeess 0 125 mA 0 5 A Max Operating Temperature Range e 4 30 C non condensing atmosphere 7 4 Warranty Life Technologies Inc warrants apparatus of its manufacture against defects in materials and workmanship under normal service for one year from the date of receipt by the purchaser This warranty excludes damages resulting from shipping misuse carelessness or neglect Life Technologies liability under the warranty is limited to the repair of such defects or the replacement of the product at its option and is subject to receipt of reasonable proof by the customer that the defect is embraced within the terms of the warranty All claims made under this warranty must be presented to Life Technologies within one year following the date of delivery of the product to the customer This warranty is in lieu of any other warranties or guarantees expressed or implied arising by law or otherwise Life Technologies makes no other warranty expressed or implied including warranties of merchantability or fitness for a particular purpose Under no circumstances shall Life Technologies be liable for damages either consequential compensatory incidental or special sounding in negligenc
15. g clips along the lower edge of the gel for sealing purposes clamp them on the side spacers Note If spring clips are not put in place during polymerization the comb will not fit tightly which increases the possibility of leaks between wells Leave the glass plates in their near horizontal position until the acrylamide has polymerized Remove the spring clips and top clamp if used and tape Carefully slide the comb from between the glass plates Rinse the top of the gel with electrophoresis buffer to remove any unpolymerized acrylamide Rinse the comb with deionized water Operating Instructions 9 If you are using a sharkstooth comb reinsert the comb between the glass plates with the teeth toward the gel Insert the comb until the teeth just make contact with the surface of the gel figure 5 Do not allow the teeth to pierce the gel A very slight indentation of the gel should be visible when the comb is properly inserted Do not slide the comb laterally after it has come in contact with the gel SLA SASASSSSSS TSS LEELA ASE S Figure 5 Sample Loading Configuration of Sharkstooth Comb 3 1 4 Pouring the Gel Using the S2 Casting Clamp The S2 Casting Clamp allows for the gel to be filled from either the top or the bottom of the assembled plate sandwich To cast the gel from the top follow the instructions below making sure that the bottom fill port is sealed 1 To fill the gel from the top us
16. g connections must be fully seated to prevent potential sparking and fire hazards Turn on the power supply and set the voltage or wattage to the proper setting for the gel see table 2 for recommended DC power settings for various gel thicknesses Monitor the progress of electrophoresis by following the migration of a marker dye front or by any other preferred method When electrophoresis is complete turn off the power supply and disconnect both DC power cords first from the power supply and then from the sequencing apparatus 3 2 4 Post Electrophoresis 1 Open the buffer chamber drain valve to allow the buffer in the upper chamber to drain into the covered rear chamber of the lower buffer tray Open the upper safety lid and rinse the upper buffer chamber with 25 to 50 ml of deionized water to prevent buffer crystallization in the internal drain tubing Release the integral gel clamps by loosening the screw knobs and remove the gel sandwich from the sequencing apparatus Lay the gel sandwich flat with the short glass plate on top on a piece of absorbent paper Open the lower safety lid and remove the lower buffer tray from the sequencing apparatus The buffer in the open front chamber of the tray will contain any radioactive nucleotides electrophoresed out of the gel Dispose of this liquid by pouring away from the drain hole in the covered rear chamber of the tray The buffer in the rear chamber drained from the upper chamber m
17. grease Make sure that the glass plates are firmly seated against the inner surface of the casting clamp The gel is too hot Use a lower wattage or voltage setting Check that the valve knob has been released sufficiently Force water or buffer through the drain outlet with a syringe or pipette to remove possible dried buffer residue airlock or collapse in the drain tubing Verify that the system is level Make sure the system is not in a draft or otherwise being cooled unevenly Verify that the buffer depth in both buffer trays is sufficient to provide complete even contact across the full width of the gel Verify that both electrode carriers are properly clipped in place Make sure that the spring clips are placed over the side spacers Distortion of the gel thickness will cause the dye front to run at different rates across the gel Verify that the top integral gel clamps are firmly but not overly tightened The ribs of the silicone gasket should be slightly and evenly compressed but not wrinkled First loosen the integral gel clamps a partial turn If the leaking does not stop try tightening the clamps carefully Verify that the black foam blocks are securely seated against the short glass plate before casting the gel Check the silicone gasket for tears or other damage Verify that a pinhole leak or crack has not developed in the glue joints of the upper buffer reservoir Buffer is leaking from the upper buffer chamber S
18. horesis wash the urea from the wells before loading the samples Remake sample buffer with fresh formamide Ethanol precipitate DNA with ammonium acetate and ethanol Try 35S or 33P labeled dNTP s instead of 32P labeled dNTP s Label the sequencing primer with T4 polynucleotide kinase instead of internal labeling Use a shorter exposure Use cassette that assures tight contact of gel to film Do not use an intensifying screen Load less sample Shorten exposure Do not store precast gels under electrophoresis buffer overnight in the sequencing unit Pre electrophorese gel until the current starts to drop generally gt 1 2 hour Do not electrophorese gel on constant current Use constant power to maintain gel at 45 50 C Use a surface thermometer during electrophoresis Thoroughly rinse residual siliconizing agent off glass plates Ethanol wipe the acrylamide instead of through gel before using Thoroughly wash new glass plates with a strong lab soap before using Remake sample buffer with fresh formamide Ethanol precipitate DNA with ammonium acetate and ethanol Recast gel with extra acrylamide at the top of the gel Clean the flat edge of the sharkstooth comb Insert sharkstooth comb only to the point of dimpling the surface Electrophorese with constant voltage or power not constant current Do not exceed settings that yield a gel surface temperature of 45 50 C Use a buffer gradient gel Use wedge spa
19. hurchill M E A and Kam L 1987 Methods Enzymol 155 537 Hartley J and Xu L 1994 Focus 16 52 Related Products Product Accessories Spacer Set Complete with two side spacers and 12 foam blocks 0 19 mm thick Mylar 0 35 mm thick Mylar 0 4 mm thick 0 8 mm thick 1 6 mm thick Bottom Spacers 0 4 mm thick 0 8 mm thick 1 2 mm thick 1 6 mm thick Wedge Spacer Set Complete with two 0 4 to 1 2 mm thick side spacers and 12 foam blocks Comb and Spacer Set Complete with two spacers and two Mylar 0 4 mm thick combs Spacer Foam Blocks pkg of 12 Analytical DELRIN Combs 16 tooth 0 4 mm thick 0 8 mm thick 1 6 mm thick 20 tooth 0 4 mm thick 0 8 mm thick 1 6 mm thick 32 tooth 0 4 mm thick 0 8 mm thick 1 6 mm thick Custom DELRIN Combs 14 cm Mylar Sharkstooth Comb 25 pt 0 19 mm thick pkg of 6 25 pt 0 35 mm thick pkg of 6 28 cm Mylar Sharkstooth Comb 62 pt 0 35 mm thick 14 cm Vinyl Sharkstooth Comb 25 pt 0 4 mm thick pkg of 6 28 cm Vinyl Sharkstooth Combs 50 pt 0 4 mm thick pkg of 2 14 cm Vinyl Doublefine Sharkstooth Combs 49 pt 0 4 mm thick pkg of 4 Glass Plates pair S2 Casting Clamp S2 Casting Clamp pkg of 2 Gel Sealing Tape 1 5 in x 72 yd 1 roll 10 rolls Spring Clips pkg of 12 Top Clamp Cat No 21105 309 21105 366 21108 014 31109 010 31109 028 21105 077 21105 069 21105 325 21105 051 21107 016 21105 341 21965 017 21035 043 21035 050 2103
20. ide spacers at the sides and place the short glass plate inside down on the side spacers Make sure that the foam blocks at the tops of the side spacers are snug against the top of the short glass plate and that the ends of the side spacers are 4 to 7 mm short of the bottom of the assembled plate sandwich Note Before using vinyl side spacers for the first time attach a foam block at one end of each spacer Remove the protective backing from the adhesive side of a foam block align the end of the block with the end of the side spacer and press the block firmly into place Foam blocks may be replaced as needed using the extras provided with the Model S2 Apparatus 7 Seal the sides and bottom of the assembled glass plate sandwich with gel sealing tape as shown in figure 2 Tape the sides first a b and then the bottom c Extend the tape around the bottom corners in both directions to ensure an adequate seal Apply the tape as smoothly as possible to avoid forming air channels or bubbles along the edges of the glass plates Rub the tape firmly onto glass to eliminate air channels and ensure a liquid tight seal 8 Place two accessory spring clips available separately along each side near the middle and bottom of the glass plate sandwich This will help maintain uniform gel thickness while pouring the gel It is important that the spring clips be placed over the side spacers only Clamping unsupported glass will distort the thickness of the
21. le Loading Volumes for Model S2 Apparatus Square Toothed Combs as a Function of Gel ThickneSSes cceeceeeeeeeeeeeeeeeeeeeneeeeeeeeaees 8 Warning 1 Read and carefully follow the manual instructions 2 Do not alter equipment Failure to adhere to these directions could result in personal and or laboratory hazards as well as invalidate equipment warranty GEL MIx RUNNING MATE Focus TECH LINE and the Life Technologies logo are marks of Life Technologies Inc DELRIN is a registered trademark of E I duPont de Nemours amp Co gt gt Notices to Customer 1 1 Important Information This product is authorized for laboratory research use only The product has not been qualified or found safe and effective for any human or animal diagnostic or therapeutic application Uses for other than the labeled intended use may be a violation of applicable law If the product is used in a manner not specified by the manufacturer the protection provided by the product may be impaired 1 2 Warnings 1 DANGER HIGH VOLTAGE This system requires a 1 000 to 3 000 V DC power supply for operation and is therefore a source of high voltage The power supply should have earth ground leakage protection and open circuit sensing Although equipped with a safety interlock system this equipment should always be operated with extreme caution Careless handling can result in electrical shock Never operate damaged
22. lectrophoresis apparatus with upper buffer chamber buffer chamber drain valve silicone gasket removable lower buffer tray integral gel clamps and safety lids One pair of 122 cm 48 in DC power cords One pair of glass plates one short one long One pair of 0 4 mm thick vinyl side spacers One pair of 0 35 mm thick mylar side spacers Package of four 0 4 mm thick vinyl sharkstooth combs Package of two 0 35 mm thick mylar sharkstooth combs Package of 12 adhesive backed foam blocks for side spacers One roll of gel sealing tape Instruction manual S2 Casting Clamp optional Many of these components are also available separately Refer to Chapter 6 Related Products for ordering information Safety lid Vinyl side spacer with foam block Sharkstooth comb go Long glass plate E Silicone gasket Buffer chamber drain valve Short glass plate Aluminum plate Integral gel clamp G Gel support block Safety lid DC power cords Removable lower buffer tray Figure 1 Model S2 Sequencing Gel Electrophoresis Apparatus Figure 2 Taping Glass Plates Operating Instructions This chapter provides operating instructions for the Model S2 Apparatus For a list of references containing formulations of buffers and acrylamide solutions as well as other applications information for polyacrylamide gel sequencing and fragment purificatio
23. n see Chapter 5 References Please read all of the following instructions before using the Model S2 Apparatus Review figure 1 as needed to identify features and components discussed in these instructions 3 1 Gel Casting Gels can be cast after assembling the glass plates and spacers using Gel Sealing Tape or the S2 Casting Clamp These techniques are appropriate for casting polyacrylamide gels 3 1 1 Preparation of Glass Plates Using Gel Sealing Tape 1 Select a pair of glass plates one long one short Be sure that the edges to be sealed with tape are free of nicks or chips 2 Clean the glass plates thoroughly with a nonabrasive detergent and a plastic scouring pad The cleaning solution should not leave a soap residue when rinsed thoroughly Avoid scratching the surface of the glass plates Note Never use steel wool to scrub the glass plates Rinse the glass plates thoroughly with deionized water and wipe dry If desired treat the inside acrylamide contact surface of one or both glass plates usually the short plate with a siliconizing reagent following the manufacturer s instructions 5 Immediately before assembling the glass plate sandwich rinse the inside surfaces with ethanol and wipe them dry with a lint free paper towel Avoid touching the inside surfaces with your fingers 6 Assemble the glass plate sandwich in the conventional manner place the long glass plate inside up on the bench align the vinyl s
24. ng and damage to the apparatus See Chapter 4 Troubleshooting for further information Close the upper and lower safety lids and connect the DC power cords to the sequencing apparatus and the DC power supply Warning All banana plug connections must be fully seated to prevent potential sparking and fire hazards Before loading samples pre electrophorese the gels for 15 to 45 min see table 2 for recommended DC power settings for various gel thicknesses Best results are achieved with a gel surface temperature of 50 C Warning Excessive power will cause the gel to overheat and crack the glass plates Operating Instructions Figure 6 Sharkstooth Comb Sample Loading Technique Table 2 Recommended Electrophoresis Power Settings for Different Gel Thicknesses Gel Thickness mm Watts W Volts V Current mA 0 2 55 2 200 2 600 20 35 0 4 60 1 500 1 900 30 45 0 8 65 1 100 1 200 60 90 1 6 70 600 700 95 125 awedge 85 90 1 200 70 Note Power conditions were determined for a 50 C surface temperature with 1X TBE buffer a0 4 to 1 2 mm thick 3 2 2 Loading Samples 1 Prepare the samples by heating in an appropriate loading buffer to a temperature of 90 C to 100 C for 3 to 5 min and then chilling on ice 2 At the end of the pre electrophoresis period turn off the power supply and disconnect both DC power cords first from the power supply and then from the sequencing apparatus before opening the
25. oir to the fitting and slowly fill the gel Do not interupt the filling as this may introduce bubbles into the gel solution Once the gel is filled insert the comb as before and allow the gel to polymerize 3 2 Electrophoresis 3 2 1 Pre Electrophoresis 1 Before beginning electrophoresis verify that the sequencing apparatus is in a secure and level position Place the gel sandwich in the sequencing apparatus with the short glass plate inward so that the foam blocks on the side spacers form a seal with the blue silicone gasket Rest the bottom edge of the sandwich on the ribbed gel support blocks in the lower buffer tray Secure the gel sandwich with the integral gel clamps along the sides of the sequencing apparatus Tighten the screw knobs firmly but do not overtighten Verify that the upper buffer chamber drain valve is closed and fill the upper buffer chamber with approximately 450 ml of electrophoresis buffer Make sure no buffer is leaking from the upper buffer chamber Fill the front chamber of the lower buffer tray with approximately 450 ml of electrophoresis buffer Be sure no bubbles obstruct buffer contact with the lower edge of the gel Note Do not overfill the buffer chambers or allow buffer to make contact with the electrode mounting nuts Warning No buffer should leak through or around the silicone gasket or down the side of the gel assembly Leakage may allow the upper buffer chamber to drain dry or cause sparki
26. or leaking equipment Always turn off the DC power source prior to disconnecting DC power cords from the sequencing apparatus Disconnect power cords from the power source first and then from the apparatus For maximum safety always operate this system in an isolated low traffic area not accessible to unauthorized personnel Before applying DC power to this equipment make sure that all electrical connections are secure and that power cords show no signs of damage See Chapter 7 for further information Certain reagents indicated for use in this manual are of a hazardous nature The researcher is cautioned to exercise care when handling these reagents The equipment used in these procedures e g high voltage power supplies should be used following the manufacturer s safety recommendations Overview 2 1 Description The Model S2 Sequencing Gel Electrophoresis Apparatus is designed for vertical polyacrylamide gel separations of the products of dideoxy and chemical sequencing reactions The system is useful for preparative purifications of oligonucleotides DNA footprinting procedures ribonuclease protection assays and sequence determinations of up to 250 to 350 bases This manual provides operating instructions for the Model S2 Apparatus 2 2 Components The Model S2 Apparatus is designed for easy assembly and reliable performance Please refer to figure 1 to identify the following principal features and components e E
27. tional Information 7 1 Care and Handling The components of the Model S2 Apparatus are fabricated from ABS acrylic vinyl glass silicone and aluminum As with any laboratory instrument adequate care ensures consistent and reliable performance After each use rinse buffer chambers glass plates combs and side spacers with deionized water Wipe dry with a soft cloth or paper towel or allow to air dry Whenever necessary all components may be washed gently with water and a nonabrasive detergent and rinsed and dried as above To remove grease and oils use a light application of hexane kerosene or aliphatic naphtha Never use abrasive cleaners window sprays or scouring pads to clean the components as these cause surface damage Additional cautions e Do not autoclave or dry heat sterilize the apparatus or components e Do not expose the apparatus or components to phenol acetone benzene halogenated hydrocarbon solvents or undiluted laboratory alcohols e Avoid prolonged exposure of the apparatus or components to UV light 7 2 Maintenance The following simple routine inspection and maintenance procedures will help ensure both the safety and the performance of the Model S2 Apparatus For ordering information for replacement parts refer to Chapter 6 or call the TECH LINE 1 Electrical connections e Inspect electrical connections and power cords regularly If the banana plug connectors to either the unit or the power
28. upper safety lid 3 Immediately prior to loading the samples rinse the wells of the gel with electrophoresis buffer Use a Pasteur pipette to wash away urea that has diffused into the wells Note Not rinsing the wells will prevent samples from layering properly on the gel surface 4 Using a sharkstooth comb to form shallow wells see Section 3 1 3 allows you to load samples with a micropipette or a drawn out capillary pipette figure 6 When using the sharkstooth comb provided with the Model S2 Apparatus load 2 to 3 ul of sample per well When using accessory doublefine sharkstooth combs load 1 5 to 2 ul of sample When using optional square toothed combs load samples onto the bottom of the wells with capillary pipettes To determine the sample loading volumes of Model S2 Apparatus square toothed combs relative to gel thickness see table 3 Table 3 Sample Loading Volumes for Model S2 Apparatus Square Toothed Combs as a Function of Gel Thickness Number Tooth Width Gel Thickness Capacity Well of Teeth mm mm pl 16 14 6 0 4 25 0 8 50 1 6 100 20 11 1 0 4 19 0 8 38 1 6 76 32 5 7 0 4 10 0 8 20 1 6 40 Note All loading volumes are calculated for a comb insertion depth of 5 mm Operating Instructions 3 2 3 Electrophoresis 1 After loading the samples close the upper and lower safety lids and connect the DC power cords to the sequencing apparatus and the DC power supply Warning All banana plu
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