Sequi-Gen® GT Nucleic Acid Electrophoresis Cell Instruction Manual
Contents
1. Sequi Gen GT Nucleic Acid Electrophoresis Cell Instruction Manual Catalog Numbers 165 3860 165 3861 165 3862 and 165 3863 For Technical Service Call Your Local Bio Rad Office or in the U S Call 1 800 4BIORAD 1 800 424 6723 Note To insure best performance from the Sequi Gen GT electrophoresis system become fully acquaint ed with these operating instructions before using the cell Bio Rad recommends that you first read these instructions carefully Then assemble and disassemble the cell completely without casting a gel After these preliminary steps you should be ready to cast and run a gel Bio Rad also recommends that all Sequi Gen GT components and accessories be inspected for dam age cleaned as recommended in this manual and rinsed thoroughly with distilled water before use Record the following for you records Model Catalog No Date of Delivery Warranty Period Serial No Invoice No Purchase Order No Warranty Bio Rad Laboratories warrants the Sequi Gen GT electrophoresis system against defects in materials and workmanship for 1 year If any defects occur in the instrument during this warranty peri od Bio Rad Laboratories will repair or replace the defective parts free The following defects howev er are specifically excluded 1 Defects caused by improper operation 2 Repair or modification done by anyone other t2. Precision Caster Base Precision Caster Gasket Precision Caster Syringe Precision Caster Tubing 60 cm Precision Caster Luer Tapers IPC Drain Port Tubing Connector Gel Temperature Indicator Vinyl Spacers 0 4 mm thick Vinyl Sharkstooth Comb 0 4 mm thick Leveling Bubble Instruction Manual e ra N e Fe A e e be ben Fe Fe ben Fe Fei Fei Parts come in 21 cm or 38 cm widths GT IPC and Glass Plates are 21 x 40 21 x 50 38 x 30 cm or 38 x 50 cm sizes GT Clamp Sets and Vinyl Spacers are either 30 cm 40 cm or 50 cm lengths Syringe sizes are 60 cc for 21 cm systems and 140 cc for 38 cm systems Vinyl Sharkstooth combs are 24 well 25 teeth for 21 cm units or 49 well 50 teeth for 38 cm units LN WN rs See Section 6 for information on accessories and replacement parts General Description The Sequi Gen GT DNA sequencing cell uses several innovative design features that are especially useful for DNA RNA sequencing or other nucleic acid separation applications Sequi Gen GT DNA sequencing cell features and benefits include Features Benefits Unique horizontal syringe injected gel Easy gel casting without tape grease and casting method acrylamide spills and waste Upper buffer chamber heat Provides uniform gel temperature that distribution system prevents smiling Permanently sealed upper buffer No gaskets or grease needed to provide leak chamber free electrophoresis continued on the next page 3
3. Gibson T J and Hong G F Buffer gradient gels and 35S label as an aid to rapid DNA sequence determination Proc Natl Acad Sci USA 80 3963 3965 1983 Bishop M J Software for molecular biology 1 Databases and search programs Bio Essays 1 25 27 Deininger P Approaches to rapid DNA sequence analysis Anal Biochem 135 247 263 1983 Garoff H and Ansorge W Improvements of DNA sequencing gels Anal Biochem 115 450 457 1981 Henikoff S Unidirectional digestion with exonuclease Ill creates targeted breakpoints for DNA sequencing Gene 28 351 359 1984 Hindley J DNA Sequencing Elsevier Biomed Press 1983 Lane D J Pace B Olsen G J Stahl D A Sogin M L and Pace N R Rapid determination of 16S ribosomal RNA sequences for phylogenetic analysis Proc Natl Acad Sci USA 82 6955 6959 1985 Maxam A M and Gilbert W Sequencing end labeled DNA with base specific chemical cleavages Methods in Enzymology 65 449 580 1980 Messing J New M13 vectors for cloning Methods in Enzymology recombinant DNA Techniques 101 20 79 1983 Messing J Crea R and Seeburg P H A system for shotgun DNAsequencing Nuc Acids Res 9 2871 2887 1981 Ornstein D L and Kashdan M A Sequencing DNA using 35S labeling A troubleshooting guide BioTechniques 3 476 483 1985 Sanger F Nicklen S and Coulson R DNA sequencing with chain terminating inhibitors P
4. 34 x 30 cm 34 x 50 cm Gel thickness range 0 25 0 75 mm Nominal gel volumes 0 25 mm 17 ml 21 x 40 cm 21 ml 21 x 50 cm 40 ml 38 x 30 cm 43 ml 38 x 50 cm Nominal gel volumes 0 40 mm 27 ml 21 x 40 cm 34 ml 21 x 50 cm 50 ml 38 x 30 cm 68 ml 38 x 50 cm Minimum upper buffer volumes 500 ml 21 x 40 cm 575 ml 21 x 50 cm 650 ml 38 x 30 cm 1 400 ml 38 x 50 cm Minimum lower buffer volume 350 ml Maximum lower buffer volume 500 ml Electrical Specifications Electrical Safety Certification IEC 1010 1 Rated voltage limit 3 000 volts Rated power limit 100 watts Rated temperature limit 60 C Electrical cables Rated to 3 000 volts VDC Electrical leads Rated to 3 000 volts VDC Banana plugs Rated to 3 000 volts VDC Construction Specifications GT IPC panel GT safety covers Universal base Stabilizer bar GT clamp set Glass plates Combs and spacers Electrodes IPC and base Banana plugs IPC and base Electrical cables Electrical leads Precision caster base Tubing Luer taper Gasket Syringe Drain port connector Injected molded polycarbonate Injected molded polycarbonate Injected molded polycarbonate Injected molded polycarbonate PVC clamp body Protruded G10 polyester glass cam shaft Polycarbonate insulated stainless steel rod Chemically tempered 4 8 mm float glass Plastic or machined vinyl see Sections 2 7 and 6 1 Platinum 0 25 mm diameter Gold plated stainless ste
5. 38 x 50 cm 2 165 3866 GT Universal Base 165 3801 Stabilizer Bar 165 3867 GT Safety Covers with cables 21 cm 165 3868 GT Safety Covers with cables 38 cm 165 3875 GT Clamp Set 30 cm 165 3876 GT Clamp Set 40 cm 165 3877 GT Clamp Set 50 cm 165 3878 Precision Caster Assembly 21 cm 26 165 3879 Precision Caster Assembly 38 cm 165 3886 Precision Caster Base 21 cm 165 3887 Precision Caster Base 38 cm 165 3888 Precision Caster Gasket 21 cm 165 3889 Precision Caster Gasket 38 cm 165 3891 Precision Caster Syringe 60 cc 165 3892 Precision Caster Syringe 140 cc 165 3893 Precision Caster Tubing 60 cm 165 3894 Precision Caster Luer Taper 4 165 3895 Drain Port Connector 2 165 3720 Gel Temperature Indicator 5 Vinyl Spacers 165 3812 Machined Vinyl Spacers 30 cm 0 4 mm red 165 3813 Machined Vinyl Spacers 30 cm 0 25 mm blue 165 3814 Machined Vinyl Spacers 40 cm 0 4 mm red 165 3815 Machined Vinyl Spacers 40 cm 0 25 mm blue 165 3816 Machined Vinyl Spacers 50 cm 0 4 mm red 165 3817 Machined Vinyl Spacers 50 cm 0 25 mm blue 165 3818 Machined Vinyl Spacers 30 cm 0 75 mm grey 165 3819 Machined Vinyl Spacers 40 cm 0 75 mm grey 165 3828 Machined Vinyl Spacers 50 cm 0 75 mm grey 165 3820 Machined Vinyl Wedge Spacers 40cm 0 25 0 75mm blue 165 3821 Machined Vinyl Wedge Spacers 40cm 0 4 0 1 2mm red 165 3822 Machined Vinyl Wedge Spacers 50cm 0 25 0 75mm blue 165 3823 Machined Vinyl Wed
6. Sliding glass plates or plate dropping methods always result in acrylamide spills and waste Cleaning the hazardous neurotoxin after the spills is also time consuming The precision caster allows quick and easy gel casting without acrylamide spills or waste By casting the gel with a syringe through the precision caster base gels can be poured in less than 1 minute The gel is cast with the glass plate assembly in the horizontal position Two full length clamps secure the assembly and allow attachment of the precision caster base to the bottom of the glass plate sandwich A seal between the caster gasket and the plates is created without tape or grease The gel is injected from the bottom of the glass plate sandwich via the injection port of the precision caster base and moves to the top of the glass plates as a dome shaped gel front Acrylamide spills and waste can be eliminated by controlling the flow of the gel front at the top of the glass plates Modular Assembly There are four IPC dimensions to choose from as shown in Figure 1 1 One universal base functions as the lower buffer chamber for all IPC sizes cl 21x40cm 21x50cm 38 x 30 cm 38 x 50 cm Fig 1 1 Interchangeable sizes 1 2 Specifications General Specifications Base footprint 16 x 48cm Maximum unit height 65 cm 50 cm cells 55 cm 40 cm cells 45 cm 30 cm cells IPC sizes 21 x 40 21 x 50 38 x 30 cm and 38 x 50 cm width x length Actual gel sizes 17 x 40 17 x 50
7. plunger after the gel has been cast Figure 4 8 11 Insert the comb s between the plates to the desired depth If a sharkstooth comb is used insert the flat edge of the comb no more than 5 mm past the short glass plate Clamp the comb s in place with three large metal binder clamps Fig 4 8 Syringe position for gel polymerization 16 e Alternatively prior to injecting the gel solution insert the corner of the comb to facilitate comb placement and insertion after gel casting 12 Let the gel polymerize for 30 60 minutes e After gel polymerization remove the luer taper from the precision caster base e The syringe tubing and luer taper can be cleaned of any remaining polymerized gel solution by rinsing with hot tap water followed by a distilled water rinse 13 Remove the precision caster base from the IPC assembly and clean the caster base and gasket of polymerized gel solution with tap water followed by a distilled water rinse 4 4 Preparing for Operation Adhere a gel temperature indicator onto the outside of the outer plate somewhere near the center to monitor the gel temperature during electrophoresis e Place the IPC assembly into the universal base against the back wall between the alignment tabs Insert the stabilizer bar Figure 4 9 e The stabilizer bar should slide into place with a snug fit locking the IPC to the base in a vertical position e The heads of the screws
8. Features Benefits A universal base accepts all gel dimensions including wide and narrow gel formats of various lengths Injection molded parts Chemically tempered glass plates One piece lever operated clamps Modular system allows different sized gels to be used with the same lower buffer chamber Provides years of rigorous use Resists cracking due to overheating and rough handling Conveniently and easily slides onto gel sandwich and shields edges of glass plates from operator contact Molded chambers with pour spouts or drain ports Easy and safe radioactive buffer disposal Machined vinyl spacers and sharkstooth combs Uniform thickness of combs and spacers reduces well to well leakage during sample loading Sequi Gen GT Buffer Heat Dissipation Uneven dissipation of the Joule heat produced by the gel during electrophoresis causes electrophoresis artifacts Smiling is a common artifact that develops when a gel sandwich loses heat more efficiently at the edges than in the center When a gel runs hotter in the center the electrical resistance decreases and more current flows down the center As the current flow increases the gel heats even more Thus a positive feedback loop is set up which results in the lanes near the center of the gel running hotter and therefore faster than the lanes near the edges Smiling can lead to ambiguity in reading the sequence The Sequi Gen GT cell employs natural con
9. No gel solution entering gel mold Gel solution is leaking into the precision caster base Gel solution is receding from the top of the gel Bubbles in gel Luer taper tubing or syringe orifices blocked Precision Caster Base injection port misaligned with gap between the glass plates Precision caster gasket missing Gasket hole not aligned with the injection port of the precision caster base Glass plates and gasket are not in contact Glass plates spacers and clamps are not flush at their bottom edge Entire casting assembly preci sion caster assembly and IPC assembly at too high of an incline Glass plates spacers and clamps are not flush Entire casting assembly preci sion caster assembly and IPC assembly at an incline Air bubbles injected into the mold because gel was injected too quickly Gel solution not degassed Air trapped in tubing Air bubbles trapped in syringe Air injected into the gel mold because gel volume was inadequate 22 Thoroughly clean syringe tubing and tapers of dried gel solution with warm water and mild detergent see Section 3 Readjust precision caster base so that white slit can be seen between the green glass plates while looking through the injection port see section 4 Insert gasket into base Disassemble precision caster base from the IPC assembly adjust the gasket and reassemble Disassemble and make sure the bottom edges of the clamps glass pla
10. all times during the run e Remove the comb s from between the glass plates e Thoroughly rinse the resulting well s or gel front using a syringe with a needle or disposable plastic transfer pipet catalog number 223 9911 e Ifusing a sharkstooth comb insert the comb with the teeth facing the gel front Lower the comb toward the gel surface until the teeth of the comb just touch the gel surface 5 Fill the lower buffer chamber with 350 500 ml of the running buffer Refer to Appendix 7 1 for running buffer recipes Caution Do not fill the lower chamber with more than 500 ml of buffer The lower buffer chamber holds the entire volume of the upper buffer chamber should a leak develop in the IPC Buffer levels over 500 ml will not allow the entire volume of the upper buffer chamber to be contained in the universal base 6 Attach the top and bottom safety covers and pre electrophorese the gel at normal operating voltage or power see Section 4 7 if desired to increase the gel temperature e Pre electrophoresis prior to sample loading will create a uniform gel temperature and bring the gel temperature to the recommended run temperature This will help eliminate any smile patterns from developing early in the run Note Gel electrophoresis buffers can be heated to 50 C in a microwave before adding buffer into the upper buffer chamber This will reduce the time needed to bring the gel to the appropriate run temperature before sample l
11. gradually thicker toward the bottom As thickness increases resistance voltage and DNA mobility decrease The resulting gel has bands more closely spaced at the bottom Wedge spacers allow the use of standard polyacrylamide solution and buffers No alterations to the gel solution gel casting or electrophoresis protocols are required to run DNA sequencing wedge gels 33 7 3 Gel Drying and Autoradiography The radiolabeled oligonucleotides may be visualized by a variety of techniques involving autoradiography For the best resolution and signal intensity dry DNA sequencing gels with a slab gel dryer 1 Transfer sequencing gels to a fresh sheet of filter paper Wet the gel slightly by misting the gel with deionized H O Lay the dry filter paper on top of the gel and press firmly The gel will stick to the paper Pick up the gel by lifting the filter paper carefully from one end 2 Cover the sequencing gel with plastic wrap Smooth out air bubbles and folds by rubbing with a paper towel and trim the edges to fit the slab gel dryer 3 Set Model 583 Gel Dryer to sequencing cycle 30 minutes at 80 C should suffice for drying thin low percent gels if the applied vacuum is above 28 inches of mercury or 125 torr Refer to the dryer s instruction manual for details 4 Autoradiograph the gel with high speed X ray film such as Kodak XAR and a suitable film cassette Intensifying screens are optional If S radiolabel is used the gel can
12. on the stabilizer bar should push against the front wall of the base to press the IPC clamps against the back wall of the universal base Note When first setting up your Sequi Gen GT cell adjust the screws on the stabilizer bar if the fit seems too loose or too tight turning the screws counterclockwise makes the stabilizer bar fit more tightly Too much pressure will make it difficult to insert and remove the stabilizer bar Too little pressure will result in the stabilizer bar sliding in and out of position without pressing the IPC against the back wall of the base Fig 4 9 Inserting the stabilizer bar into the universal base 17 3 To avoid buffer spills and cell tipping accidents adjust the leveling screws on the universal base as necessary e To insure that the unit will not tip over during electrophoresis make sure the leveling feet threaded rods are at least 1 cm deep into the threaded boss of the base e At this time test whether the IPC assembly is properly aligned in the universal base by attaching the top and bottom safety covers The IPC assembly may have to be shifted to the right or the left to properly attach the safety covers After this final alignment is complete remove the safety covers 4 Fill the upper buffer chamber the IPC with running buffer 1x TBE using the flared portion of the panel as a fill spout e The level of the buffer should be about 1 cm from the top of the fill spout at
13. 0 Al Before Assembly EE 10 4 2 Assembling the Glass Plate Sandwich 10 4 3 Casting E 12 4 4 Preparing for Operations ee 17 GV WEN E ie 18 4 6 Gel Electrophoresis yeee neiaie i r AR 19 AT DPis sse mbl ye er E E E E E 20 Section5 Troubleshooting Guide sesesocsesccscsosocsosocscsosscsocscsosscsosocsosossososscsossceossse 22 5 1 Operational Troubleshooter 1 22 5 2 IDNA SeqiienCime ATUA S aeons senesi es 23 Section 6 Equipment and Accessories osososososoessosocscscsesosososososososososossesesosososososo 26 6 1 Sequi Gen GT Nucleic Acid Electrophoresis Cells and Accessories 00 26 6 2 Electrophoresis Reagent 20102 30 6 3 Power Supplies and Slab Gel Dryers ssssssssseesesesesrsrsrsesssrstsestsrererserersrsrsrsrersreree 31 6 4 DNA Template Purification Sequencing and Cloning Droducrs eee 31 63 Laeud Handing eege 32 Section7 Appendix A Applications sesssosscsosscscsocscsccscecescsosocsosocsososscsossesossceossse 32 7 1 DNA Sequencing Checklet AAA 32 K2 Standard Gel Protocol 2 eteiedeteiedeteieieteiedebagedeteieieteeieteie dia fedete deier iteiedebegeieieb die 33 7 3 Gel Drying Autoradiography Au 34 7 4 Applications for Sequi Gen GT Nucleic Acid Electrophoresis Cell 34 7 5 Suggested Reading AA 35 Section 1 General Information 1 1 Introduction to the Sequi Gen GT Nucleic Acid Sequencing Cell The Sequi Gen GT cell is a modular electrophoresis cell capable of separating nucleic acid
14. 0 VDC Maximum operating power 100 Watts Electrical current to the Sequi Gen GT cell enters the unit through the top and bottom safety covers providing a safety interlock to the user Current flow to the cell is broken when either safety cover is removed Do not attempt to circumvent this safety interlock Always turn the power supply off while working with the sequencing cell when the safety covers are not connected No user serviceable parts are contained in this apparatus To insure electrical safety do not attempt to service this apparatus Caution Arcing Arcing within an electrophoresis cell is represented by sparks smoke or charred surfaces created when an electrical short has developed Arcing can occur if the buffer level drops below the recommended height if there is buffer leakage or if loose electrical connections exists If arcing is detected during electrophoresis immediately remove the source of electrical current i e turn off the power supply Always use a power supply that is capable of detecting electrical conditions that may cause accidental electrical shock or damage to the apparatus The PowerPac 3000 power supply contains safety features such as arc no load overload rapid change in resistance and ground leak detection capabilities that will reduce the chance of accidental electrical shock and damage to the electrophoresis cell Before every use inspect all plastic parts glass plates all electrical
15. 02 Flamingo Valley Singapore 1545 Phone 65 4432529 Fax 65 4421667 Spain Bio Rad Laboratories S A Avda Valdelaparra 3 Pol Ind Alcobendas E 28100 Alcobendas Madrid Phone 91 661 70 85 Fax 91 661 96 98 Sweden Bio Rad Laboratories AB Gardsvagen 7D Box 1276 S 171 24 Solna Phone 46 0 8 735 83 00 Fax 46 0 8 735 54 60 Switzerland Bio Rad Laboratories AG Kanalstrasse 17 Postfach CH 8152 Glattbrugg Phone 01 809 55 55 Fax 01 809 55 00 United Kingdom Bio Rad Laboratories Ltd Bio Rad House Maylands Avenue Hemel Hempstead Herts HP2 7TD Free Phone 0800 181134 Fax 01442 259118 SIG 051995 Printed in USA
16. 21 x 50 cm 0 40 mm 50 60 W 21 x 50 cm 0 75 mm 55 65 W 21 x 50 cm 0 25 0 75 mm wedge 55 65 W 21 x 50 cm 0 4 1 2 mm wedge 55 65 W continued on the next page 19 Table 4 3 continued Sequi Gen GT Gel Recommended Cell Size Thickness Power Setting 38 x 30 cm 0 25 mm 70 75 W 38 x 30 cm 0 40 mm 70 75 W 38 x 30 cm 0 75 mm 70 75 W 38 x 30 cm 0 25 0 75 mm 70 75 W 38 x 30 cm 0 40 1 20 mm 70 75 W 38 x 50 cm 0 25 mm 70 80 W 38 x 50 cm 0 40 mm 75 85 W 38 x 50 cm 0 75 mm 75 85 W 38 x 50 cm 0 25 0 75 mm wedge 75 85 W 38 x 50 cm 0 40 1 2 mm wedge 75 85 W Important Never allow the gel temperature to exceed 60 C Severe damage to the glass or adhesive bond may result Caution Periodically check the level of the upper buffer to make sure that it is at least 1 cm above the short glass plate 2 Continue gel electrophoresis until the desired fragment size separation is achieved Typically gel electrophoresis times are monitored by observing the dye front mobility of either the bromophenol blue fast blue or xylene cyanol slow blue during the course of elec trophoresis Fragment and dye front mobility as a function of polyacrylamide percentage are shown in Table 4 4 below and should be used as a guide for gel electrophoresis monitoring Table 4 4 Migration of Single stranded DNA in Denaturing Polyacrylamide Gels in Relation to Dye Marker Gel Migration Polyacrylamide Bromophenol Gel Percentage Blue Xylene Cyanol 5
17. 35 bases 130 bases 6 26 bases 106 bases 8 19 bases 75 bases 10 12 bases 55 bases From Ausubel F M et al Current Protocols in Molecular Biology Greene and Wiley 1993 4 7 Disassembly 1 When the desired dye front mobility has been achieved turn off the power supply and remove both safety covers e The upper buffer chamber can be partially emptied by inserting the drain port connector and any attached tubing into the drain port on the IPC A click will be heard when the drain port tubing connector has been properly inserted Figure 4 10 e Buffer will begin to drain from the IPC immediately after the connector is inserted into the drain port 20 Fig 4 10 Inserting the drain port connector for upper buffer chamber drainage 2 After the upper buffer chamber is emptied to the level of the drain port pull out the stabilizer bar and remove the IPC assembly Blot the bottom edge of the IPC assembly onto absorbent paper before removing it to a nearby sink Carefully pour the remaining upper buffer out of the IPC assembly into a sink Slowly and carefully pour the lower buffer contained in the universal base into the appropriate sink or container Caution Never store buffers in an IPC Never add buffer to an IPC unless the clamps are in place The lever clamps provide the necessary force to keep the static head pressure of the upper buffer from straining the adhesive bond Remove t
18. 5 ammonium persulfate solution catalog number 161 0700 e Choose the appropriate syringe and tubing assembly tubing and luer taper provided with the precision caster Insert the luer taper into the one end of the tubing Secure the other end of the tubing onto the luer end of the syringe When the gel solution has degassed add 25 APS and TEMED catalog number 161 0800 in the recommended amounts see Section 7 2 e Swirl the solution gently to mix e Slowly pull the required gel volume into the syringe see Table 4 1 e Tap air bubbles to the top of the syringe luer end and gently force them out If bub bles are inadvertently introduced into the tubing pinch the portion of the tubing where the bubbles exist while forcing some of the gel solution out This should allow the bubble to exit the tubing with the gel solution 14 9 When all air bubbles are removed from the tubing place the luer taper into the injection port of the precision caster base Figure 4 6 Tighten the luer taper fitting in place on the injection port of the precision caster base and begin to slowly inject the gel solution Slow and even pressure on the syringe plunger will insure uniform gel casting with no bubbles Figure 4 7 Fig 4 7 Injecting gel solution into glass plate sandwich 15 Note on Gel Bubble Formation The following injection times from the bottom of IPC to the top were found to result in bubble free gel
19. Box Sterilized 223 9480 EZ Micro Test Tube 1 5 ml 500 Box 223 9503 EZ Micro Test Tube 0 5 ml 500 Box Section 7 Appendix 7 1 DNA Sequencing Checklist For DNA sequencing you will need the following buffers reagents and equipment 1 DNA sequencing samples suitably labeled see Section 6 4 2 10x TBE buffer 108 g Tris base 55 g boric acid 9 3 g Na EDTA H_O in 1 liter deionized H O autoclave The pH of this solution should be 8 3 without adjustment see Section 6 2 3 Acrylamide stock solution 30 A For low percent gels 4 10 Prepare a 30 stock solution 19 1 Acrylamide Bis 28 5 g Acrylamide 1 5 g Bis Acrylamide 30 0 g Total up to 100 ml deionized H O see Section 6 2 For medium percent gels 8 16 Prepare a 30 stock solution 29 1 Acrylamide Bis 29 0 g Acrylamide 1 0 g Bis Acrylamide 30 0 g Total up to 100 ml deionized H O see Section 6 2 For high percent gels 12 20 Prepare a 40 stock solution 37 5 1 Acrylamide Bis 38 96 g Acrylamide 1 04 g Bis Acrylamide 40 0 g Total up to 100 ml deionized HO see Section 6 2 4 TEMED see section 6 2 32 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Ammonium Persulfate 25 stock solution 0 25 g in 1 ml distilled H2O in a microfuge tube Make fresh daily see Section 6 2 A constant power or constant voltage power supply see Section 6 3 Slab gel dryer see Section 6 3 Table top m
20. Plastic components made from self extinguishing material e Full length clamps to shield user from edges of glass plates e Chemically tempered glass plates that significantly reduce glass plate breaking due to overheating and routine heating and cooling e Noexposed metallic parts e Pour spout in lower buffer chamber allows radioactive buffer to be easily and safely poured for disposal Important This apparatus meets I E C 1010 1 safety standards Sequi Gen GT systems are safe to use when operated in accordance with the instructions This instrument should not be modified in any way Alteration of this instrument will e Void the manufacturer s warranty e Void the IEC1010 1 safety certification e Create a potential safety hazard IEC1010 1 is an internationally accepted electrical safety standard for laboratory instruments 1 Bio Rad is not responsible for any injury or damage caused by the use of this instrument for purposes other than those for which it is intended or by modifications to the instrument not performed by Bio Rad or an authorized agent Power to the Sequi Gen GT cell is supplied by an external DC power supply This power supply must be ground isolated in such a way that the DC voltage output floats with respect to ground The recommended power supply for this apparatus is the PowerPac 3000 power supply The maximum specified operating parameters for the Sequi Gen GT cell are Maximum operating voltage 3 00
21. Premixed reagents and buffers are also available and offer convenience time savings and reproducible results Each reagent and buffer is purified to meet rigorous quality control standards See Section 6 2 for ordering information 2 3 Electrical Path Both electrode wires are positioned near the bottom of the gel The upper buffer carries the current from the cathode up to the top of the plates near the fill spout where the gel is exposed The lower buffer contacts the gel at the bottom edge of the plates in the standard fashion See Figure 2 3 Fill spout Upper buffer Polycarbonate panel Bonded glass plate Gel IPC drain port Silicone adhesive bond Lower buffer Fig 2 3 Electrical path through IPC Integral Plate Chamber to lower buffer reservoir Section 3 Cleaning and Maintenance 3 1 Cleaning and Siliconizing Plates Important To insure bubble free gels using the Sequi Gen GT precision caster the glass plates must be thoroughly cleaned and the outer long glass plate siliconized or coated before each use 1 Clean both Sequi Gen GT glass plates IPC and outer plates thoroughly before each use e Carefully place the plate into the sink and rinse with warm water e Pour powdered lab detergent Alconox Alconox Inc or Micro International Products into a gloved hand and add sufficient water to make a paste e Apply the paste and scrub the entire glass surface w
22. Vacuum Pump Trap tubing and connectors 165 1753 Model 583 Gel Drying System 220 240 V includes Model 583 Gel Dryer Vacuum Pump Trap tubing and connectors 165 1745 Model 583 Slab Gel Dryer 100 120 V 165 1746 Model 583 Slab Gel Dryer 220 240 V 165 0959 Sequencing Gel Filter Paper 35 x 45 cm 25 sheets 165 0962 Filter Paper Backing 35 x 45 cm 25 sheets 165 0963 Cellophane Membrane Backing 35 x 45 50 sheets 6 4 DNA Template Purification Sequencing and Cloning Products Catalog Number Product Description DNA Template Purification 732 6100 Quantum Prep Plasmid Miniprep Kit 100 preps DNA Template Sequencing 170 3407 170 3414 170 3409 Bst Premixed Standard Sequencing Kit 50 reactions Bst Premixed 7 deaza dGTP Sequencing Kit 50 reactions Bst adjustable Ratio Sequencing Kit 50 reactions DNA Mutagenesis 170 3580 170 3581 Muta Gene M13 In Vitro Mutagenesis Kit 25 reactions Muta Gene Phagemid In Vitro Mutagenesis Kit 25 reactions 31 6 5 Liquid Handling Catalog Number Product Description 223 9911 Seque Pro Capillary Tips 200 Box 223 9912 Seque Pro Capillary Tips 200 Box autoclaved 223 9314 MTP 39 Pipet Tips 960 Box 223 9319 MTP 39 S Pipet Tips 960 Box Sterilized 211 2001 Xcluda Aerosol Barrier Pipet Tips 0 5 10 ul 960 Box Sterilized 211 2006 Xcluda Aerosol Barrier Pipet Tips 5 20 ul 960 Box Sterilized 211 2016 Xcluda Aerosol Barrier Pipet Tips 20 200 960
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24. be left on the outer glass plate and fixed in 1 liter of 10 acetic acid 10 methanol for 15 minutes This removes hygroscopic urea The gel may then be dried on filter paper Removal of plastic wrap before autoradiography is important because S is a weak beta emitter Autoradiography of S labeled fragments typically requires 1 3 days However we have found the fixative step unnecessary even when sequencing with S 7 4 Nucleic Acid Separation Applications for the Sequi Gen GT Electrophoresis System Several other nucleic acid separation techniques requiring single nucleotide resolution can be conducted using the Sequi Gen GT systems Below is a comprehensive list Refer to Sambrook J Fritsch E F and Maniatas T Molecular Cloning A Laboratory Manual Second Edition Cold Spring Harbor Laboratory Press 1989 or Ausubel F M et al Current Protocols in Molecular Biology Greene Publishing Associates and Wiley Interscience 1987 for more information and protocols e Microsatellite Analysis e Single Strand Conformational Polymorphism SSCP studies e Heteroduplex analysis e DNA footprinting e DNA fingerprinting e RNase protection assays e SI nuclease mapping e Primer extension studies e DNA Protein binding studies gel shift assays e Oligonucleotide analysis 34 7 5 Suggested Reading Bankier A T and Barrell B G Shotgun DNA Sequencing Techniques in Life Sciences Vol B5 Elsevier 1983 Biggin M D
25. cables jacks and receptors for loose connections cracks charring or corrosion Do not use any part that is cracked chipped charred or corroded These parts may cause arcing Contact your Bio Rad representative before using a part that may cause buffer leaking or arcing Warning Never allow the gel to exceed 60 C Excessive heat may crack the plates or cause the silicon bond of the IPC to deteriorate Make sure that the upper and lower buffer chambers are filled with buffer during electrophoresis Do not allow the buffer level to drop below the level of the short glass plate of the upper buffer chamber or below the bottom of the IPC assembly in the lower buffer chamber at any time Certain solvents and cleaning agents should be avoided with this unit Refer to Section 3 2 for compatible solvents reagents and cleaning agents Definition of Symbols A A Caution risk of electrical shock Caution refer to accompanying documents 2 System Components Each Sequi Gen GT system comes with the components listed in Table 1 1 Check your unit to be sure all items are present Note any damage to the unit which may have occurred during shipping Notify Bio Rad Laboratories if any items are missing or damaged Table 1 1 Sequi Gen GT System Components Item Quantity GT Universal Base Stabilizer Bar GT Safety Covers GT IPC with bonded inner short glass plate Outer long Glass Plate GT Clamp Set left and right clamp
26. cm of Wells Width mm Separation mm Volume pl Plastic Sharkstooth Comb 165 3700 0 40 15 24 6 1 None 7 3 165 3702 0 40 15 48 3 1 None 3 7 165 3701 0 25 15 24 6 1 None 4 5 165 3703 0 25 15 48 3 1 None 2 3 Plastic Well forming Comb 165 3684 0 40 14 16 6 7 2 4 42 5 165 3685 0 25 14 16 6 7 2 4 26 5 165 3686 0 40 14 20 4 9 2 4 31 0 165 3687 0 25 14 20 4 9 2 4 19 5 165 3688 0 40 14 36 2 4 1 6 3 6 165 3689 0 25 14 36 2 4 1 6 2 3 165 3692 0 40 31 32 7 4 2 4 47 0 165 3693 0 25 31 32 7 4 2 4 29 5 165 3694 0 40 31 44 4 8 2 4 30 0 165 3695 0 25 31 44 4 8 2 4 18 5 165 3696 0 40 31 60 3 6 1 6 5 5 165 3697 0 25 31 60 3 6 1 6 3 4 165 3698 0 40 31 80 2 3 1 6 3 5 165 3699 0 25 31 80 2 3 1 6 2 2 MP Plastic Well forming Combs 165 3848 0 40 15 34 165 3849 0 40 30 68 165 3850 0 75 15 34 165 3851 0 75 30 68 Maximum well volumes are calculated values based on an assumed well height Well height will vary with each user The well volumes indicated will vary from run to run and user to user 29 6 2 Electrophoresis Reagents Catalog Number Product Description Electrophoresis Buffers and Gel Reagents 161 5100 PAGE Reagent Starter Kit 1 includes Acrylamide 100 g Bis 5 g TEMED 5 ml Ammonium Persulfate 10 g Premixed Acrylamide Solutions 161 0154 30 Acrylamide Bis Solution 19 1 500 ml 161 0155 30 Acrylamide Bis Solution 19 1 2 x 500 ml 161 0144 40 Acrylamide Bis Solution 19 1 500 ml 161 0145 40 Acrylamide Bis Sol
27. e and gel buffers Pour new gel with better lanes Check sample and gel buffers e Check sample and gel buffers 3 Total Signal Artifacts Condition Probable Causes Solutions Preventions Large horizontal exposed areas of film High molecular weight area distorted on film Fuzzy bands bands smeared resolution problems Autoradiogram shows large black spots or radiating patterns Film sticks to dried gel Blank autoradiogram Buffer contamination with label e Molarity or pH anomaly in gel causing dehydration gel shrinking and bubbles located near the top of the gel Improper sample loading Hydrolyzed gel matrix e Tonic contaminants in gel Polymerization problem e Improper gel temperature e Wet plastic wrap or wet gel e Static electricity sparks exposed film during handling e Gel not completely dried e Hygroscopic urea has bound water Sample dependent problem e Autoradiography problem 25 Clean IPC and buffer containers remake buffers e Gel run too hot gel buffer hydrolyzed gel buffer not made up correctly or upper buffer degraded Refer to Section 3 5 Don t run gel above 55 C e Use only electrophoresis grade reagents check purity e TEMED or APS concentration too high e Pre running gel may result in better resolution e Refer to Section 7 1 and 7 2 for protocols Refer to bulletin 1156 e Use Gel Temperature Indicator 50 C is usually high e
28. e was allowed to diffuse into gel too long before electrophoresis Loading wells not straight Sample molarity too low rela tive to gel e Sharkstooth comb inserted too far into the gel Loading wells not straight or clean Bubble in gel e Sample molarity too high Sample molarity too low Sample dependent artifact e Check sample and gel buffers e Sample dependent artifact Probable Causes e Allow gel to polymerize more completely before removing comb e Check sample and gel buffers e Reduce sample load volume e Load a set or two at a time Pour new gel with better lanes e Check sample and gel buffers e Touch the top of the gel with the teeth e Allow gel to polymerize more completely before removing comb Pour new gel without bubbles e Check sample and gel buffers e Check sample and gel buffers Solutions Preventions Smiling within sets Frowning within sets Funneling within sets Non continuous vertical register Band spacing compressed within a set Film exposure differences within aset Loading wells not straight within set e Molarity problems in the sam ples of the set Loading wells not straight Molarity problems in the sam ples of the set e Sample molarity too high or contamination e Sample dependent artifact e Autoradiographic artifact e Sample dependent artifact e Sample dependent artifact 24 Pour new gel with better lanes e Check sampl
29. el 5 08 cm length Dual 20 AWG tinned copper wire cable Flame retardant polyurethane insulation jacket Polyurethane insulated nickel silver 2 95 cm length Injection Molded Polycarbonate Polyurethane 3 2 mm internal diameter 4 8 mm outer diameter Polypropylene 3 2 mm internal diameter Silicon Foam Sponge Polypropylene 60 cc or 140 cc Polypropylene quick coupling assembly 3 2 mm internal flow diameter Section 2 Description of Major Parts 2 1 Sequi Gen GT Parts See Figures 2 1 and 2 2 for Sequi Gen GT part identification Sharkstooth Comb Syringe _ m Leveling Bubble Syringe Tubing Es GT Lever Clamps Luer Taper Precision Caster Base Injection Port Precision Caster Cam Peg Fig 2 1 Sequi Gen GT gel casting parts 6 GT Lever Clamp GT Top Safety Cover IPC Integral Plate Chamber GT Lever Clamp Stabilizer Bar GT Bottom Safety Cover GT Base gt Leveling Feet Fig 2 2 Sequi Gen GT nucleic acid electrophoresis cell 2 2 Gel Reagents and Electrophoresis Buffers For most DNA sequencing or nucleic acid separations a 19 1 acrylamide bis solution is required A 1x TBE Tris boric acid and EDTA solution is the preferred electrophoresis buffer Reproducibility is affected by the quality of the gel and buffer reagents A full line of high quality polyacrylamide gel reagents and nucleic acid electrophoresis buffers is available from Bio Rad
30. ge Spacers 50cm 0 4 1 2mm red Clear Plastic Spacers 165 3710 Plastic Spacers 40cm 0 4mm 10 165 3711 Plastic Spacers 40cm 0 25mm 10 165 3712 Plastic Spacers 50cm 0 4mm 10 165 3713 Plastic Spacers 50cm 0 25mm 10 165 3714 Plastic Spacers 80cm 0 4mm 10 165 3715 Plastic Spacers 80cm 0 25mm 10 165 3716 Plastic Spacers 100cm 0 4mm 10 165 3717 Plastic Spacers 100cm 0 25mm 10 tapers tubing and syringe 0 40 mm vinyl sharkstooth comb and spacers gel temperature indicator leveling bubble drain port All Sequi Gen GT systems include GT IPC assembly IPC and bonded inner glass plate outer glass plate and clamp set GT universal base GT safety covers with cables stabilizer bar precision caster assembly precision caster base gasket tubing luer connector and instruction manual t All Sequi Gen GT PowerPac 3000 systems include the appropriate Sequi Gen GT system described above PowerPac 3000 power supply PowerPac temperature probe and PowerPac instruction manual 27 Machined Vinyl Combs Vinyl sharkstooth combs and spacers are machined to maintain a uniform and precise thickness throughout the length of each spacer and comb and between all vinyl spacers and combs All spacers and sharkstooth combs are color coded based on thickness Blue 0 25 mm red 0 4 mm and gray 0 75 mm spacers are available in 30 40 and 50 cm lengths Blue and red sharkstooth combs are available in 15 and 30 cm len
31. gs upward Adjust the caster base either up or down until the space between the green glass plates can be seen and is in the middle of the injection port hole e While securing the precision caster base in place with one hand turn the cam pegs back to their original position to secure the base to the bottom of the IPC assembly 13 Fig 4 5 Correct alignment of precision caster base with glass plate sandwich 6 Lay the IPC assembly and attached precision caster base flat on a bench with the IPC panel drain port facing up and the long edges of the clamps running parallel with the edge of the benchtop e The most even pouring can be obtained by insuring that the assembly is level on the benchtop Failure to level the assembly may result in gel leakage A leveling bubble is provided to facilitate leveling the IPC assembly Props approximately 2 cm will be required at the top of the IPC to level the unit for casting The unit is now ready for gel casting An alternative to the use of props is to cast the gel with the precision caster positioned off the edge of the lab bench Note If casting a 38 x 50 cm IPC place the 38 x 50 cm IPC assembly at an incline with the top of the apparatus approximately 4 5 cm higher than the bottom The bottom of the apparatus contains the attached precision caster base After the gel is cast level the assembly for gel polymerization While the gel solution is degassing prepare a fresh 2
32. gths and a wide range of well formats including multichannel pipet microplate compatible MP combs for high throughput applications Catalog Comb Comb Number Well Well Maximum Well Number Thickness mm Length cm of Wells Width mm Separation mm Volume pl Machined Vinyl Sharkstooth Comb 165 3830 0 25 15 24 6 1 None 4 5 165 3831 0 25 15 36 4 1 None 3 0 165 3832 0 25 15 48 3 1 None 2 3 165 3833 0 25 30 48 6 1 None 4 5 165 3834 0 25 30 72 4 1 None 3 0 165 3835 0 25 30 96 3 1 None 2 3 165 3836 0 40 15 24 6 1 None 7 3 165 3837 0 40 15 36 4 1 None 5 0 165 3838 0 40 15 48 3 1 None 3 7 165 3839 0 40 30 48 6 1 None 7 3 165 3840 0 40 30 72 4 1 None 5 0 165 3841 0 40 30 96 3 1 None 3 7 MP Vinyl Sharkstooth Combs 165 3842 0 25 15 34 None 165 3843 0 25 30 68 None 165 3844 0 25 30 100 None 165 3845 0 40 15 34 None 165 3846 0 40 30 68 None 165 3847 0 40 30 100 None Maximum well volumes are calculated values based on an assumed well height Well height will vary with each user The well volumes indicated will vary from run to run and user to user 28 Clear Plastic Combs and Spacers Well forming combs are 14 cm and 31 cm wide All plastic well forming combs sharkstooth combs and spacers are made from inert plastic which does not catalyze or inhibit polymerization Thus the combs are easy to remove without damaging the sample loading wells Catalog Comb Comb Number Well Well Maximum Well Number Thickness mm Length
33. h warm water and wipe any polymerized acrylamide off the clamping surfaces Drain the banana plug mounts at the top of the clamps and wipe the clamping surfaces dry before each use Do not use organic solvents to clean the clamps Section 4 Operating Instructions 4 1 Before Assembly 1 Thoroughly clean all parts as described in Section 3 Caution Certain solvents and cleaning agents should be avoided Refer to Section 3 2 for compatible cleaning agents 2 Depending on the size of the Sequi Gen GT IPC make up the appropriate amount of electrode buffer typically 1x TBE from Table 4 1 Table 4 1 Electrode Buffer Volumes IPC Size Total Buffer Required Upper Lower 21x 40cm 850 ml 500 ml 350 ml 21x 50cm 925 ml 575 ml 350 ml 38 x 30 cm 1 000 ml 650 ml 350 ml 38 x 50 cm 1 750 ml 1 400 ml 350 ml 4 2 Assembling the Glass Plate Sandwich After the Sequi Gen GT components have been washed and the glass plates siliconized or coated assemble the Sequi Gen GT apparatus Always wear gloves while handling the glass plates during assembly to avoid fingerprints on the glass plates Fingerprints will cause bubbles to form during gel casting Important Before assembling the Sequi Gen GT cell inspect all plastic parts glass plates electrical cables jacks and receptors for loose connections cracks chips charring or corrosion Do not use any part that is damaged These parts may cause buffer leaks or arcing 1 Clean and siliconi
34. han Bio Rad Laboratories or an authorized agent 3 Use of fittings or other spare parts supplied by anyone other than Bio Rad Laboratories 4 Damage caused by accident or misuse 5 Damage caused by disaster 6 Corrosion due to use of improper solvent or sample This warrant does not apply to parts listed below 1 Platinum wire 2 Glass plates For any inquiry or request for repair service contact Bio Rad Laboratories after confirming the model and serial number of your instrument Table of Contents Page Warranty Information csscsscsscsssssssssssscsssssssescscssessessesssassesseses Inside Front Cover Section 1 General Information c ssiciisssocsscesesspsvessocedeossovedtastonsscessacesavbeasbonsinessevedvesbers 1 1 1 Introduction to Sequi Gen GT DNA Electrophoresis Cell 1 HD EE 5 Section 2 Description of Major Parts sssssssscssssssssssssssccssssessesssessssessesssessessoes 6 2 1 Sequi Gen GT PariS e euE E E EE S ENE eye EE OR EEE RNR 6 2 2 Gel Reagents and Electrophoresis Buffers AAA 7 2 3 Electrical Path ee Sege Ee EE 8 Section 3 Cleaning and Maintenance ccscsccscsscssssessessscssssescesssessecsessesssessessoss 8 3 1 Cleaning and Siliconizing Pilates 8 3 2 Cleaning Plastic Parts 2c 2 dotssed dovcsetssucsesdedeesssssoscsutesers subsdovescdudecesods suresetetetccotedoesestes 9 Section 4 Operating INstruction ccscsscsssssssssssssssssssssssssssssssssesssessessessesssessesses 1
35. he clamps from the IPC assembly by first pulling the levers away from the IPC and then sliding the clamps off the IPC assembly e Place the Sequi Gen GT cell flat on a bench with the outer glass plate facing up e Carefully separate the glass plates by pulling up gently near the top of the outer plate e After the plates begin separating carefully determine to which plate the gel is sticking the gel should stick to the short inner bonded glass plate on the IPC When the gel is secured onto one glass plate carefully place a piece of filter paper catalog number 165 0921 onto the gel surface Press firmly on the filter paper to make the gel adhere Trim around the filter paper with a razor blade or scissors to remove any excess gel e Remove the filter paper and gel by lifting up one end of the filter paper and carefully peeling the gel off the plate e Place the gel filter paper side down on the bench and cover it with a piece of plastic wrap Use a razor blade or scissors to trim away any excess plastic wrap The gel is now ready for drying autoradiography and interpretation of results Refer to Section 7 3 for gel drying and autoradiography procedures 21 Section 5 Troubleshooting Guide 5 1 Operational Troubleshooter The following table summarizes possible Sequi Gen GT operational difficulties probable causes and solutions Refer to Sections 3 and 4 for recommended procedures Problem Probable Causes Solution
36. icro centrifuge Gel loading syringe e g Hamilton 701 SN 28 Gauge 1 25 inch needle 1 5 ml microcentrifuge tubes see Section 6 5 Adjustable pipettors e g Pipetman P 20 P 200 P 1000 Balance Plastic wrap Pipette tips autoclaved see Section 6 5 Waterbath or Temp Block at 95 C X ray film and cassettes dark room facilities Filter Paper see Section 6 3 Siliconizing solution or glass coating solution Geiger Counter Ice bucket 7 2 Standard Gel Protocol The following protocol is for a standard 7 M urea 5 polyacrylamide gel for DNA sequencing See Section 4 for additional information on gel casting sample loading and gel electrophoresis For ordering information on gel reagent and electrophoresis buffers see Section 6 l Combine 63 g of urea 15 ml of 10x TBE and 25 ml of 30 acrylamide stock solution Bring the volume to 150 ml with distilled water low heat may be required to dissolve the urea but do not boil Filter the solution through a 0 45 micron mesh filter optional Then degas under strong vacuum 5 15 minutes to remove dissolved oxygen Add 150 ul TEMED and 150 ul 25 ammonium persulfate or one microliter of each reagent for every milliliter of gel solution prior to gel casting Cast the gel according to procedures in Section 4 Note Wedge spacers see Section 6 1 increase the number of readable bases per lane in a sequencing gel The use of wedge spacers results in a gel which becomes
37. igned or flush adjust the alignment by loosening the clamps and move clamps glass plates and spacers into alignment Figure 4 3 e Tighten the clamps by moving the levers back down towards the IPC after the assembly is flush oe Fig 4 3 Alignment of glass plate sandwich 6 To avoid incompatibility problems between combs and spacers after the gel is cast check the fit of the combs in the assembled Sequi Gen GT cell by trying to place them between the plates e Ifthe combs clearly will not fit between the plates without damaging the comb try a different comb Optimally combs should demonstrate slight resistance to being placed between the glass plates 4 3 Casting the Gel Section 7 1 contains a checklist of required items for DNA sequencing Polyacrylamide is a hazardous chemical and neurotoxin Always wear gloves lab coat and safety glasses while working with polyacrylamide 1 Prepare the gel solution described in Section 7 1 and 7 2 e Degas the gel solution for 5 15 minutes under a strong vacuum 2 26 in Hg to insure reproducible gel porosity Table 4 2 Required Gel Volumes Using the Precision Caster Assembly IPC 025mm 0 4mm 075mm 0 25 0 75mm 0 40 1 2 mm Size spacers spacers spacers wedge spacer wedge spacer 21 x 40cm 25 ml 35 ml 70 ml 50 ml 60 ml 21 x 50 cm 30 ml 45 ml 90 ml 65 ml 85 ml 38 x 30 cm 40 ml 50 ml 90 ml 38 x 50 cm 55 ml 85 ml 170 ml 120 ml 140 ml 12 2 Place the p
38. incorrectly Gel hydrolyzed more conductive Gel too hot or cold Neither plate siliconized Both plates siliconized Plates unclean Outer plate pried off too quickly 5 2 DNA Sequencing Artifacts Refill upper buffer chamber e Caution Monitor run Refill upper buffer chamber e Polymerization problem Stop the run Electrical hazard IPC needs replacement Refill upper buffer chamber 350 ml is minimum 500 ml is maximum Insert comb minimum distance e Rinse comb gel with buffer before pulling out comb Pull comb out slowly e Refer to Bulletin 1156 e Check buffers e Remake gel run gel cooler Run gel at 50 C e Siliconize outer plate according to Section 3 1 e Separate plates slowly Electrophoretic artifacts are described below A DNA sequencing artifact may be defined as any non ideal graphic pattern on the X ray film that reduces your confidence in reading or interpreting a sequence from that film There are three types of DNA sequencing artifacts e Template dependent artifacts e Electrophoretic artifacts e Autoradiographic or data acquisition artifacts Template specific artifacts are caused by biological or chemical phenomena and relate to issues beyond the scope of this manual Each sequencing method has its own set of potential sequence specific artifacts Section 7 5 contains references that discuss sequence specific artifacts The basic premise for reading a DNA sequence
39. is that each band on the film exists in a vertical register that corresponds to one base in the sequence Non ideal patterns caused by problems in the three categories above may interfere with the accurate determination of DNA sequences The following is a guideline for description and analysis of artifacts in DNA sequencing gels especially electrophoretic ones Electrophoretic Artifacts There are many sources of electrophoretic artifacts To simplify the task of defining an artifact we use a systematic description of electrophoretic artifacts dividing all of the possible patterns into three hierarchical sub categories e Lane local artifacts e Set template local artifacts 23 e Total signal artifacts Many artifacts appear in more than one sub category For example smile patterns can exist in lane local or in total signal situations or both but rarely appear in set local It is important to distinguish the extent and location of each artifact before trying to analyze or troubleshoot the anomalous pattern 1 Lane local artifacts Condition Probable Causes Solutions Preventions Smiling bands Frowning bands Complex curvy bands Funneling or lane narrowing Lane widening Variations in signal intensity along the lane s Band spacing compressed or stretched 2 Set local artifacts Condition Loading wells not straight Sample molarity too high relative to gel Sample overloaded e Sampl
40. ith a gloved hand using circular motions 8 2 e Rinse off all of the detergent with warm water e Rinse with deionized water e Wipe the cleaned plate with a large lint free tissue to dry Inspect the plates carefully for pieces of detergent dried polyacrylamide or other particles Rewash if necessary Perform siliconizing under a fume hood to reduce the hazard from breathing silanizing reagent Alternatively several non toxic non corrosive glass plate coating solutions are commercially available We recommend siliconizing or coating only the outer long plate so that when the plates are separated the gel sticks to the IPC bound glass plate e Use a glass Pasteur pipette to dispense 2 ml of the silanizing reagent onto the front plate Coat the plate completely and evenly by spreading the silanizing reagent on the plate surface with a large lint free tissue using a motion that travels from the top to the bottom of the plate Caution Do not siliconize the IPC plate unless hexane heptane or water is used as a solvent in the silanizing reagent Other organic solvents will craze or damage the IPC plastic and weaken the adhesive bond e Never heat an IPC in an oven Severe damage will result to the adhesive bond Use siliconizing compounds that react or cure at room temperature Note If the gels will be fixed or stained the IPC short plate should be siliconized or coated since its immersion into fixing or staining soluti
41. nough e Re expose with dry Saran Wrap e Do not rub film prior to placing or removing film e Dry gels longer Remove urea by soaking gel in methanol acetic acid before drying Section 6 Equipment and Accessories 6 1 Sequi Gen GT Nucleic Acid Electrophoresis Cells and Accessories Catalog Number Product Description 165 3860 Sequi Gen GT System 21 x 40 cm 165 3861 Sequi Gen GT System 21 x 50 cm 165 3862 Sequi Gen GT System 38 x 30 cm 165 3863 Sequi Gen GT System 38 x 50 cm 165 3802 Sequi Gen GT PowerPac 3000 System 21 x 40 cm 100 120 V 165 3805 Sequi Gen GT PowerPac 3000 System 21 x 40 cm 220 240 V 165 3803 Sequi Gen GT PowerPac 3000 System 21 x 50 cm 100 120 V 165 3806 Sequi Gen GT PowerPac 3000 System 21 x 50 cm 220 240 V 165 3810 Sequi Gen GT PowerPac 3000 System 38 x 30 cm 100 120 V 165 3811 Sequi Gen GT PowerPac 3000 System 38 x 30 cm 220 240 V 165 3804 Sequi Gen GT PowerPac 3000 System 38 x 50 cm 100 120 V 165 3807 Sequi Gen GT PowerPac 3000 System 38 x 50 cm 220 240 V 165 3870 GT IPC Assembly 21 x 40 cm 165 3871 GT IPC Assembly 21 x 50 cm 165 3872 GT IPC Assembly 38 x 30 cm 165 3873 GT IPC Assembly 38 x 50 cm 165 3880 GT IPC 21x 40cm 165 3881 GT IPC 21 x 50cm 165 3882 GT IPC 38x 30cm 165 3883 GT IPC 38 x 50 cm 165 3644 Outer Glass Plates 38 x 30 cm 2 165 3882 Outer Glass Plates 21 x 40 cm 2 165 3646 Outer Glass Plates 21 x 50 cm 2 165 3649 Outer Glass Plates
42. oading and will greatly reduce pre electrophoresis time Warning The upper buffer level may drop slightly due to evaporation as the system becomes warmer Make sure that the upper chamber is always filled with buffer during electrophoresis Do not allow the buffer level to drop below the level of the notched shorter IPC glass plate at any time during electrophoresis as this may cause arcing and cell damage Additionally never allow the gel to exceed 60 C under any circumstance This excessive heat may crack the plates or cause the IPC glass bond to deteriorate 4 5 Loading the Gel 1 Turn off the power supply and remove the top safety cover e Rinse the well s with a syringe with needle or disposable plastic transfer pipet catalog number 223 9911 to remove urea before applying the samples to the gel 18 2 Load samples on the gel see Table 2 1 for recommended sample loading volumes for all Bio Rad combs e Samples may be applied with a 5 ul Hamilton syringe or a pipettor fitted with gel loading tips use Bio Rad catalog number 223 9911 e Syringe loading requires rinsing the needle between samples e Be sure to reconnect the upper safety cover before turning on the power supply Note Sample loading is the key to high resolution gels e Rinse wells thoroughly before sample loading begins e Deposit samples directly on the gel surface e Electrophorese the samples into the gel soon after loading every 4 lane set
43. ons is not recommended Prior to assembling the plates apply a small amount of ethanol to each plate and rub to dryness with a tissue Using the same tissue clean the spacers 3 2 Cleaning Sequi Gen GT Components 1 2 Rinse the universal base buffer chamber stabilizer bar combs spacers and precision caster base gasket syringe and tubing assembly with a mild detergent solution in warm water Use a soft bristled brush or sponge to remove polyacrylamide gel pieces Note Do not snag or break the electrode wire in the universal base while cleaning Rinse thoroughly with warm water and air dry Compatible Cleaning Agents for Polycarbonate Parts Chemically compatible cleaners must be used to ensure long life of parts These include e Aqueous solutions of soaps and mild detergents e Organic solvents e Hexane e Aliphatic hydrocarbons e Alcohols e Methanol e Ethanol e Isopropyl alcohol e Dilute acids Caution Do not touch plastic molded parts with solvents that contain chlorinated hydrocarbons or aromatic hydrocarbons e g carbon tetrachloride toluene methyl ethyl ketone acetone Do not use abrasive or highly alkaline cleaners on the polycarbonate plastic IPC panel The glass may be cleaned with abrasive or strong alkaline detergents if adequate care is taken to avoid contact with the plastic panel Do not soak plastic parts in detergents more than 30 minutes Cleaning the Clamps Rinse the clamps wit
44. recision caster base on the bench with its open cavity facing up Place the gray precision caster gasket into the base The cam pegs in the precision caster must be pulled out to accommodate the apparatus Note If the gasket is wet remove any remaining water from the gasket by squeezing it with a paper towel 3 Place the bottom edge of the IPC assembly into the precision caster base with the bottom edge of the assembly resting against the gray gasket of the precision caster base 4 When the IPC assembly is seated in the caster base use the cam pegs to connect the base to the clamps Figure 4 4 e Push each cam peg into the corresponding hole on the clamp with the lever in the up position Slight downward pressure applied to the top of the IPC assembly may be required to engage each cam peg Fig 4 4 Attaching precision caster base to IPC assembly 5 When both pegs are engaged turn them evenly until moderate resistance is felt or the han dles of the cam pegs are perpendicular to the benchtop This action causes the precision caster base to fit tightly against the plate assembly e Lay the IPC assembly flat on the benchtop with the precision caster base facing toward you e Look through the injection port of the base If the precision caster has been attached properly a space should be seen between the two green glass plates Figure 4 5 e Ifthe space cannot be seen loosen the caster base by rotating the cam pe
45. roc Natl Acad Sci USA 74 5463 5467 1977 Schreier P H and Cortese R J A fast and simple method for sequencing DNA cloned in the single stranded bacteriophage M13 J Mol Biol 169 172 1979 Staden R Automation of the computer handling of gel reading data produced by the shotgun method of DNA sequencing Nucleic Acids Res 10 4731 4751 1982 Tabor S and Richardson C C Proc Natl Acad Sci USA 84 4767 4771 1987 Yanisch Perron C Viera J and Messing J Improved M13 phage cloning vectors and host strains Nucleotide sequences of the M13mp18 and pUC19 vectors Gene 33 103 119 1985 35 36 BIO RAD Molecular Bioscience Group 2000 Alfred Nobel Drive Hercules California 94547 Telephone 510 741 1000 Fax 510 741 1060 400 0069 Rev B Bio Rad Laboratories Eastern Regional Office 85A Marcus Dr Melville New York 11747 Phone 516 756 2575 Fax 516 756 2594 Australia Bio Rad Laboratories Pty Limited Unit 11 112 118 Talavera Rd P O Box 371 North Ryde NSW 2113 Phone 02 805 5000 Fax 02 805 1920 Austria Bio Rad Laboratories Ges m b H Auhofstrasse 78D 1130 Wien Phone 1 877 89 01 Fax 1 876 56 29 Belgium Bio Rad Laboratories S A N V Begoniastraat 5 9810 Nazareth Eke Phone 09 385 55 11 Fax 09 385 65 54 Canada Bio Rad Laboratories Canada Ltd 5671 McAdam Road Mississauga Ontario L4Z 1N9 Phone 905 712 2771 Fax 905 712 2990 China Bio R
46. s for 50 cm gels with 0 4 mm spacers between 40 45 seconds for 50 cm gels with 0 25 spacers between 50 65 seconds Injection times of 10 seconds or less can result in bubble formation in the gel Bubbles can form at the gel front because of soiled areas or uneven siliconization or coating of the glass plates To achieve bubble free gels thoroughly clean both plates and siliconize the outer glass plate before each use If bubbles begin to form at the gel front hard tapping on top of the IPC assembly above the bubble formation while slowly injecting the gel solution should eliminate the bubble Alternatively the comb end of the IPC assembly can be momentarily lifted at an angle to facilitate elimination 10 Continue to slowly inject the gel solution until the gel solution emerges a few centimeters from the top of the notched shorter glass plate across the entire width of the gel Important If pouring a 38 x 50 cm IPC remove the support that created an incline and lay the unit level on the benchtop use the Leveling Bubble provided An additional 2 cm support will be needed to level the IPC assembly Some users find it convenient to use two 1 5 ml tube racks as props When the gel is past the short plate lay the syringe on top of IPC assembly until gel polymerization is complete Do not remove the luer taper from the precision caster base injection port or the gel solution will drain out of the plates Do not adjust the syringe
47. s to reduce sample diffusion and enhance band resolution 4 6 Gel Electrophoresis 1 Make sure both safety covers are in place e Apply the voltage by pressing the Start or Run button on the power supply e Verify that current is flowing note bubbles forming at the cathode wire in the IPC and that all electrical connections are solid Running the gel with constant power watts will result in a constant gel temperature during the run and reproducible gel electrophoresis Power conditions for DNA sequencing gels are usually dictated by gel running temper ature Run sequencing gels at 50 C for best results Refer to the following table for typical power watts settings that result in 50 C runs These settings are only guidelines optimal settings for gels should be determined empir ically Use a temperature indicator one is included with this unit to monitor running temperatures If the temperature goes above 55 C reduce the power output of the supply Alternatively use a power supply with temperature control functions PowerPac 3000 with temperature probe to monitor and control gel temperature Table 4 3 Approximate Power Watts Settings for Operating Sequi Gen GT Cells Sequi Gen GT Gel Recommended Cell Size Thickness Power Setting 21 x 40cm 0 25 mm 35 45 W 21 x 40 cm 0 40 mm 40 50 W 21 x 40 cm 0 75 mm 45 55 W 21 x 40 cm 0 25 0 75 mm wedge 45 55 W 21 x 40 cm 0 4 1 2 mm wedge 45 55 W 21 x 50 cm 0 25 mm 45 55 W
48. s with single base pair resolution using a vertical slab gel format This manual tells you how to operate and care for your new Sequi Gen GT cell Read Sections 1 through 3 before attempting to assemble the cell The remainder of the manual gives you detailed procedures a troubleshooting guide and parts lists The Sequi Gen GT cell employs a simple design that provides maximum resolution with high reproducibility while eliminating the temperature artifacts which often occur in sequenc ing gels Some of the unique features of this sequencing cell are the gel casting method durable construction modular components and ease of operation which make this the most advanced DNA sequencing cell available Note This manual contains instructions for the Sequi Gen GT electrophoresis systems only Prior to the release of the Sequi Gen GT systems Bio Rad supplied two similar sequenc ing electrophoresis cell systems the original Sequi Gen cell and the Sequi Gen II cell This manual does not provide information on these systems Contact your Bio Rad represen tatives for information on the original Sequi Gen and the Sequi Gen II systems US Patent number 4 663 015 issued to Bio Rad Laboratories Safety AN A The Sequi Gen GT cell has safety features to protect the operator from injury These features include e Interlocking safety lids to prevent high voltage buffer shock e Permanently sealed upper buffer chamber to prevent leaks and arcing e
49. tes and spacers are flush Disassemble and make sure the bottom edges of the clamps glass plates and spacers are flush Lower the casting assembly or completely level the assembly Disassemble and make sure the bottom edges of the clamps glass plates and spacers are flush Level the casting assembly Refer to injection rate recom mendations under Note on Gel Bubble Formation Section 4 3 Degas gel before casting Remove tubing before drawing gel solution into the syringe then attach tubing and gently push gel solution through tubing Draw gel solution into the syringe barrel slowly to avoid introducing bubbles on the side of the barrel See Section 4 4 Table 4 2 for suggested gel volumes Condition Probable Causes Solutions Preventions Upper buffer level drops too fast during run Sparks at the top of the gel Sparks in lower chamber Well forming loading wells deform when comb pulled out Unexpected power conditions Gel sticks to both plates when opening sandwich Normal consequence of IPC plastic bowing slightly as it heats up Spacers leaking out the sides of the gel Buffer leaks down between gel and spacers Bond failure Chamber leaking Sparks or burn marks in adhesive Upper buffer level dropped below minimum level Lower buffer level too low or too high Comb inserted too far Gel polymerized too long dried out Comb pulled out too quickly Gel not polymerized Buffers made
50. ution 19 1 500 ml Premixed Acrylamide Bis Powders 161 0120 Acrylamide Bis 19 1 30 g 161 0123 Acrylamide Bis 19 1 150 g Crosslinkers and Catalysts 161 0200 Bis 5g 161 0201 Bis 50 g 161 0800 TEMED 5ml 161 0801 TEMED 50 ml 161 0700 Ammonium Persulfate 10 g Premixed Buffers 161 0741 Premixed 10x TBE Extended Range 1 L 161 0758 Premixed 10x TBE Extended Range 6 x 1 L 161 0733 Premixed 10x Tris Boric Acid EDTA TBE 1 L 161 0756 Premixed 10x Tris Boric Acid EDTA TBE 6 x 1 L Powders and Reagents 161 0100 Acrylamide 99 9 100 g 161 0101 Acrylamide 99 9 500 g 161 0107 Acrylamide 99 9 1 kg 161 0103 Acrylamide 99 9 2 kg 161 0108 Acrylamide 99 9 5 kg 161 0730 Urea 250 g 161 0731 Urea 1 kg 161 0716 Tris 500g 161 0719 Tris 1 kg 161 0750 Boric Acid 500 g 161 0751 Boric Acid 1 kg 161 0728 EDTA 100g 161 0729 EDTA 500 g 1 Hazardous shipping charges may apply 2 Store at 4 C 3 For a longer shelf life store desiccated at room temperature 30 6 3 Power Supplies and Slab Gel Dryers Catalog Number Product Description Power Supplies 165 5056 PowerPac 3000 Power Supply 110 120 V 165 5057 PowerPac 3000 Power Supply 220 240 V 165 5059 PowerPac 3000 Power Supply with Temperature Probe 110 120 V 165 5060 PowerPac 3000 Power Supply with Temperature Probe 220 240 V Slab Gel Dryers 165 1752 Model 583 Gel Drying System 110 120 V includes Model 583 Gel Dryer
51. vection and conduction of the upper buffer to distribute heat evenly The problems of uneven heat dissipation are avoided Complicated expensive thermostatic plates are not necessary A thin transparent upper buffer chamber called an IPC Integral Plate Chamber acts as a heat sink across the full area of the gel Convection occurs any time a slight temperature gradient develops mixing the buffer and heat to prevent smile patterns from developing Convection is the most effective way to distribute heat evenly The upper buffer dampens temperature fluctuations in the gel and adds to the reproducibility of each run The contact between the buffer and the gel plate is direct and uniform Thermal and physical stresses are reduced The sample loading wells are always at the same temperature as the gel resulting in fewer re annealing problems Bubbles of gas generated by electrolysis along the cathode rise through the buffer These bubbles also help to prevent temperature gradients from forming by stirring the upper buffer while rising to the top of the IPC chamber Sequi Gen GT Gel Casting Because of their large size casting sequencing gels has traditionally been extremely problematic Taping the bottom or sides of the glass plate sandwich is time consuming and does not always result in a perfect seal Thus vacuum grease is required to seal corners and edges The user must then wrestle with the gel mold in order to pour the gel correctly
52. ze the glass plates as instructed in Section 3 1 2 Place the IPC flat on the bench with glass plate facing upward Figure 4 1 e Position one spacer along each long edge of the IPC glass plate The bottom edges of the spacer and the glass plate should be flush and the long edge of the spacer should be next to the plastic lip of the IPC panel 10 Fig 4 1 Assembling glass plate sandwich 3 Place the front outer long glass plate onto the IPC and spacers with the siliconized or coated surface facing down e With both hands stand the IPC glass plate sandwich on the benchtop with the outer glass plate facing away from you e Allow the glass plates and spacers to touch the benchtop to temporarily align the assembly for gel casting 4 Slide the clamps over the IPC assembly e The levers of the clamps should be on the IPC panel side of the assembly and need to be facing away from the unit perpendicular to the IPC panel for the clamps to slide easily onto the assembly Secure the clamps to the IPC glass plate sandwich by moving the levers toward the IPC panel Figure 4 2 Fig 4 2 Attaching full length lever clamps 11 5 Lay the IPC assembly on the benchtop with the IPC panel drain port side facing up e Check the alignment of the glass plates spacers and clamps The bottom of the glass plates spacers and clamps should be flush If either glass plate spacer or clamp is not properly al
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