Bio-Rad User`s Guide for Gel Boxes
Contents
1. 161 0097 Zeta Probe GT Genomic Tested Positively Charged Nylon Blotting Membrane sheets 15 x 15 cm 15 Zeta Probe GT Genomic Tested Positively Charged Nylon Blotting Membrane sheets 15 x 20 cm 15 Zeta Probe GT Genomic Tested Positively Charged Nylon Blotting Membrane sheets 20 x 20 cm 15 Zeta Probe GT Genomic Tested Positively Charged Nylon Blotting Membrane sheets 20 x 25 cm 3 Zeta Probe GT Genomic Tested Positively Charged Nylon Blotting Membrane roll 30 cm x 3 3 m 1 Zeta Probe GT Genomic Tested Positively Charged Nylon Blotting Membrane roll 20 cm x 3 3 m 1 Supported Nitrocellulose Membrane 0 45 micron sheets 7 x 8 4 cm 10 Supported Nitrocellulose Membrane 0 45 micron sheets 10 x 15 cm 10 Supported Nitrocellulose Membrane 0 45 micron sheets 15 x 15 cm 10 Supported Nitrocellulose Membrane 0 45 micron sheets 20 x 20 cm 10 Supported Nitrocellulose Membrane 0 45 micron roll 30 cm x 3 m 1 Supported Nitrocellulose Membrane 0 20 micron sheets 7 x 8 4 cm 10 Supported Nitrocellulose Membrane 0 20 micron sheets 15 x 15 cm 10 Supported Nitrocellulose Membrane 0 20 micron roll 30 cm x 3 m 1 Vacuum Blotting Apparatus 165 5000 165 5001 165 5002 170 3940 170 3948 170 3949 165 5031 165 5032 165 5033 165 5034 Model 785 Vacuum Blotter Model 785 Vacuum Blotter System 120 VAC Model 785 Vacuum Blotter System 220 240 VAC Semi Dry Transfer2. 21 BIO RAD Life Science Group 2000 Alfred Nobel Drive Hercules California 94547 Telephone 510 741 1000 Fax 510 741 5800 M1704400 REV A Bio Rad Laboratories Australia Bio Rad Laboratories Pty Limited Block Y Unit 1 Regents Park Industrial Estate 391 Park Road Regents Park NSW 2143 Phone 02 9414 2800 Fax 02 9914 2888 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 LAZ 1N9 Phone 905 712 2771 Fax 905 712 2990 China Bio Rad Laboratories 14 Zhi Chun Road Hai Dian District Beijing 100088 e Phone 01 2046622 Fax 01 2051876 Denmark Bio Rad Laboratories Symbion Science Park Fruebjergvej 3 DK 2100 Copenhagen e Phone 39 17 9947 Fax 39 27 1698 Finland Bio Rad Laboratories Business Center L nsikeskus Pihat rm 1A SF 02240 Espoo e Phone 90 804 2200 Fax 90 804 1100 France Bio Rad S A 94 96 rue Victor Hugo B P 220 94 203 Ivry Sur Seine Cedex e Phone 1 49 60 68 34 Fax 1 46 71 24 67 Germany Bio Rad Laboratories GmbH HeidemannstraBe 164 D 80939 M nchen Postfach 450133 D 80901 M nchen e Phone 089 31884 0 Fax 089 31884 100 India Bio Rad Laboratories C 248 Defence Colony New Delhi 110 024 Phone 91 11 461 0103 Fax
3. 170 4420 170 4421 170 4422 170 4423 170 4424 170 4425 170 4416 170 4426 170 4430 170 4431 170 4422 170 4432 170 4433 170 4434 170 4435 170 4436 Sub Cell GT Base Sub Cell GT Safety Lid with Cables Gel Caster Sub Cell GT Electrode Anode red Sub Cell GT Electrode Cathode black Sub Cell GT Gel Casting Gates GT UVTP Gel Tray 15 x 10 cm GT UVTP Gel Tray 15 x 15 cm GT UVTP Gel Tray 15 x 20 cm GT UVTP Gel Tray 15 x 25 cm Wide Mini Sub Cell GT Systems Wide Mini Sub Cell GT Base Wide Mini Sub Cell GT Safety Lid with Cables Mini Gel Caster Wide Mini Sub Cell GT Electrode Anode red Wide Mini Sub Cell GT Electrode Cathode black Wide Mini Sub Cell GT Gel Casting Gates GT UVTP Gel Tray 15 x 10 cm GT UVTP Gel Tray 15 x 7 cm Mini Sub Cell GT Systems Mini Sub Cell GT Base Mini Sub Cell GT Safety Lid with Cables Mini Gel Caster Mini Sub Cell GT Electrode Anode Red Mini Sub Cell GT Electrode Cathode Black Mini Sub Cell GT Gel Casting Gates GT UVTP Gel Tray 7 x 10 cm GT UVTP Gel Tray 7 x 7 cm 16 Sub Cell Systems Combs Fixed Height Combs For Sub Cell GT and Wide Mini Sub Cell GT Systems Catalog Well Thickness Well Width Well Volume Number Number mm mm Capacity ul 170 4440 1 1 50 106 43 800 0 170 4441 2 1 50 50 29 377 0 170 4442 4 1 50 26 42 200 0 170 4443 10 0 75 9 87 37 0 170 4444 10 1 50 9 87 74 0 170 4445 15 0 75 5 52 20 7 170 4446 15 1 50 5 52 41 4 170 4447 20
4. Sub Cell GT Agarose Gel Electrophoresis Systems Instruction Manual Catalog Numbers 170 4401 to 170 4406 170 4481 to 170 4486 For Technical Service Call Your Local Bio Rad Office or in the U S Call 1 800 4BIORAD 1 800 424 6723 Warranty Bio Rad Laboratories warrants the Sub Cell GT Wide Mini Sub cell GT and Mini Sub cell GT electrophoresis systems against defects in materials and workmanship for year If any defects occur in the instrument during this warranty period Bio Rad Laboratories will repair or replace the defective parts free The following defects however are specifically excluded 1 Defects caused by improper operation 2 Repair or modification done by anyone other than 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 warranty does not apply to parts listed below 1 Platinum Electrode Wires To insure the best performance from the Sub Cell GT electrophoresis systems become fully acquainted with these operating instructions before use Bio Rad recommends that you first read these instructions carefully Assemble and disassemble the unit 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 Sub Cell
5. 0 75 4 84 18 2 170 4448 20 1 50 4 84 36 4 170 4449 30 1 50 2 69 20 2 Multi channel Pipet Compatible MP Fixed Height Combs For Sub Cell GT and Wide Mini Sub Cell GT Systems Catalog Well Thickness Well Width Well Volume Number Number mm mm Capacity ul 170 4450 10 0 75 5 82 21 8 170 4451 10 1 50 5 82 43 6 170 4452 14 0 75 5 82 21 8 170 4453 14 1 50 5 82 43 6 170 4454 18 0 75 2 91 10 9 170 4455 18 1 50 2 91 21 8 170 4456 26 0 75 2 91 10 9 170 4457 26 1 50 2 91 21 8 Adjustable Height Combs For Sub Cell GT and Wide Mini Sub Cell GT Systems Adjustable height combs require a comb holder catalog 170 4320 Catalog Well Thickness Well Width Well Volume Number Number mm mm Capacity ul 170 4328 1 1 50 106 43 800 0 170 4345 2 1 50 50 29 377 0 170 4325 10 0 75 9 87 37 0 170 4326 10 1 50 9 87 74 0 170 4323 15 0 75 5 52 20 7 170 4324 15 1 50 5 52 41 4 170 4321 20 0 75 4 84 18 2 170 4322 20 1 50 4 84 36 4 170 4344 30 1 50 2 69 20 2 17 Fixed Height Combs for Mini Sub Cell GT Catalog Well Thickness Well Width Well Volume Number Number mm mm Capacity ul 170 4460 1 1 50 43 43 325 7 170 4461 2 1 50 20 32 152 4 170 4462 8 0 75 5 54 20 8 170 4463 8 1 50 5 54 41 6 170 4464 15 0 75 2 59 9 7 170 4465 15 1 50 2 59 19 4 Adjustable Height Combs for Mini Sub Cell GT Adjustable height combs require a comb holder catalog 170 4331 Catalog Well Thickness Well Width Well Volume Numbe
6. 1000 UV Gel Documentation System PC 220 240 VAC Gel Doc 1000 UV Gel Documentation System Mac 100 VAC Gel Doc 1000 UV Gel Documentation System Mac 120 VAC Gel Doc 1000 UV Gel Documentation System Mac 220 240 VAC 20 Section 7 References 1 Sambrook Fritsch and Maniatis Molecular Cloning A Laboratory Manual Second Edition Cold Spring Harbor Laboratory Press 1989 Current Protocols in Molecular Biology Greene Publishing Associates and Wiley Interscience 1989 Additional Reading SOP oo SOY Ur Be SOS Kopchick J J Cullen B R and Stacey D W Anal Biochem 115 419 1981 Southern E Methods in Enzymol 68 152 1979 The Bio Rad Silver Stain Bulletin 1089 Bio Rad Laboratories Hercules CA Bittner M Kupferer P and Morris C F Anal Biochem 102 459 1980 Bio Rad Trans Blot Cell Operation Instructions Bulletin 1082 Bio Rad Laboratories Hercules CA Winberg G and Hammarskjold M L Nucleic Acids Res 8 253 1980 Jytatekadze T V Axelrod V D Gorbulev V G Belzhelarskaya S N and Vartikyan R M Anal Biochem 100 129 1979 10 Dretzen G Bellard M Sassone Corsi P and Chambon P Anal Biochem 112 295 1981 The Polymerase Chain Reaction PCR process is covered by patents owned by Hoffmann LaRoche Use of the PCR process requires a license EC 1010 1 is an internationally accepted electrical safety standard for laboratory instruments
7. 91 11 461 0765 Italy Bio Rad Laboratories Sr Via Cellini 18 A 20090 Segrate Milano e Phone 02 21609 1 Fax 02 21609 399 Japan Nippon Bio Rad Laboratories 7 18 Higashi Nippori 5 Chome Arakawa ku Tokyo 116 e Phone 03 5811 6270 Fax 03 5811 6272 The Netherlands Bio Rad Laboratories B V Fokkerstraat 10 3905 KV Veenendaal Phone 0318 540666 Fax 0318 542216 New Zealand Bio Rad Laboratories Pty Ltd P O Box 100 051 North Shore Mail Centre Auckland 10 Phone 09 443 3099 Fax 09 443 3097 Pacific Bio Rad Laboratories Unit 1111 11 F New Kowloon Plaza 38 Tai Kok Tsui Road Tai Kok Tsui Kowloon Hong Kong e Phone 7893300 Fax 7891257 Singapore Bio Rad Laboratories Singapore Ltd 221 Henderson Rd 05 19 Henderson Building Singapore 0315 e Phone 65 272 9877 Fax 65 273 4835 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 G rdsv gen 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 e Phone 01 809 55 55 e 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 020996 Printed in USA
8. Cells Trans Blot SD Semi Dry Electrophoresis Transfer Cell Trans Blot SD System 100 120 VAC Trans Blot SD System 220 240 VAC UV Crosslinking Chamber GS Gene Linker UV Chamber 120 VAC GS Gene Linker UV Chamber 220 VAC GS Gene Linker UV Chamber 240 VAC GS Gene Linker UV Chamber 100 VAC 19 Gel Reagents and Standards 162 0019 162 0133 162 0126 170 8200 170 8210 170 8220 170 3470 170 3465 161 0404 161 0423 161 0433 161 0733 161 0743 161 0719 161 0751 161 0729 170 3742 170 3746 170 3747 170 7520 170 7521 170 7522 170 7525 170 7526 170 7527 Low Melt Preparative Grade Agarose 100 g Molecular Biology Certified Agarose 500 g High Strength Analytical Grade Agarose 500 g AmpliSize DNA Size Standard 50 2 000 bp DNA Size Standard 1 4 2 Kb ladder DNA Size Standard 0 7 8 4 Kb DNA Size Standard A Hind III DNA Size Standard pBR322 AVa II Eco RI Bromophenol Blue 10 g Xylene Cyanole FF 25 g Ethidium Bromide Solution 10 ml 10 mg ml Electrophoresis Buffers 10x Tris Boric Acid EDTA TBE 1 I 50x Tris Acetic Acid EDTA TAE 1 Tris 1 kg Boric Acid 1 kg EDTA 500 g DNA Gel Image Analysis and Documentation Systems Standard Polaroid Documentation System 120 VAC Standard Polaroid Documentation System 100 VAC Standard Polaroid Documentation System 220 240 VAC Gel Doc 1000 UV Gel Documentation System PC 100 VAC Gel Doc 1000 UV Gel Documentation System PC 120 VAC Gel Doc
9. GT system components and accessories be inspected for damage cleaned as recommended in this manual and rinsed thoroughly with distilled water before use Record the following for your records Model Catalog No Date of Delivery Warranty Period Serial No Invoice No Purchase Order No For any inquiry or request for repair service contact Bio Rad Laboratories after confirming the model and serial number of your instrument Section 1 1 1 1 2 1 3 1 4 Section 2 2 1 2 2 2 3 2 4 2 5 Section 3 Section 4 4 1 4 2 4 3 4 4 4 5 Section 5 Section 6 6 1 6 2 6 3 Section 7 Table of Contents Page General Information ccssccssssssscsccsesssssssscssesssessssersssessesesesseseressereees 1 riede tee e EE 1 NY sce ae naw ea sea nan Mest Pind wea we nau EE 1 System oppe eet 2 ee ee E A Operating INStructiONs s icisessssersessssrscnsssvesssssseassesssessvcsacsessessensssesssseesensseesee 4 DNA Gel Preparation EE SANA RR EE hohe DAN ANA RAHA NG NE Baas 4 Casting Agarose EE 6 Electrophoresis nnus oo nono n eirene b erapep pepe p o a Ope bb 8 Nucleic Acid Staining and Visualization eese 9 Note on Blotting eee one t emot et o e E OE 10 Gel and Electrophoresis Reagent Preparation 10 Care and Maintenance eere eerte sees esee sesenta tato stata enata enses enean enas 11 Cleaning Sub Cell GT Components
10. gel strength gels Section 6 Product Information 6 1 Sub Cell GT Systems Catalog Number Product Description 170 4401 Sub Cell GT System with 15 x 10 cm tray 170 4402 Sub Cell GT System with 15 x 15 cm tray 170 4403 Sub Cell GT System with 15 x 20 cm tray 170 4404 Sub Cell GT System with 15 x 25 cm tray 170 4481 Sub Cell GT System with 15 x 10 cm tray and gel caster 170 4482 Sub Cell GT System with 15 x 15 cm tray and gel caster 170 4483 Sub Cell GT System with 15 x 20 cm tray and gel caster 170 4484 Sub Cell GT System with 15 x 25 cm tray and gel caster 170 4405 Wide Mini Sub Cell GT System 170 4485 Wide Mini Sub Cell GT System with gel caster 170 4406 Mini Sub Cell GT System 170 4486 Mini Sub Cell GT System with gel caster Sub Cell GT PowerPac 300 Power Supply Systems 165 4349 Sub Cell GT PowerPac 300 System 100 120 V 165 4350 Sub Cell GT PowerPac 300 System 220 240 V 165 4348 Wide Mini Sub Cell GT PowerPac 300 System 100 120 V 165 4351 Wide Mini Sub Cell GT PowerPac 300 System 220 240 V 165 4347 Mini Sub Cell GT PowerPac 300 System 100 120 V 165 4352 Mini Sub Cell GT PowerPac 300 System 220 240 V All Sub Cell GT PowerPac 300 systems come with 15 x 15 cm UVTP tray and gel caster 15 6 2 Sub Cell GT System Accessories Catalog Number Product Description Sub Cell GT Systems 170 4410 170 4411 170 4412 170 4413 170 4414 170 4415 170 4416 170 4417 170 4418 170 4419
11. is usually conducted with either Tris Acetate EDTA TAE or Tris Boric Acid EDTA TBE While TAE provides faster electrophoretic migra tion of linear DNA and better resolution of supercoiled DNA TBE buffers have a stronger buffering capacity for longer or higher voltage electrophoresis runs Bio Rad offers premixed 50x TAE and 10x TBE buffers for use with the Sub Cell GT systems RNA formaldehyde gels require a MOPS 3 N morpholino propanesulfonic acid electrophoresis buffer 10 1x Tris Acetate EDTA TAE 40 mM tris pH 7 6 20 mM acetic acid and 1 mM EDTA 50x Stock 1 liter dissolve in 600 ml distilled water 242 g tris base FW 121 57 1 ml glacial acetic acid 100 ml 0 5 M EDTA pH 8 0 Fill to a final volume of 1 liter with distilled water 1x Tris Boric Acid EDTA TBE 89 mM tris pH 7 6 89 mM boric acid 2mM EDTA 10x Stock 1 liter dissolve in 600 ml distilled water 108 g tris base FW 121 55 g boric acid FW 61 8 40 ml 0 5 M EDTA pH 8 0 Fill to a final volume of 1 liter with distilled water 1x MOPS Buffer RNA Gels 0 02 M MOPS 3 N morpholino propanesulfonic acid pH 7 0 8 mM sodium acetate 1 mM EDTA pH 8 0 5x Stock 1 liter dissolve in 600 ml DEPC treated distilled water 20 6 g MOPS 13 3 ml 3 M sodium acetate DEPC treated pH 7 4 10 ml 0 5 M EDTA DEPC treated pH 8 0 Hill to a final volume of 1 liter with DEPC treated distilled water Caution DEPC is a suspected
12. 15 cm UVTP tray for casting in the base Slide the gel casting gates into the slots at opposite ends of the base stage Insure the gates are evenly seated in the slots and the gates uniformly contact all edges of the UVTP tray The weight of the gates provides a tight seal to prevent any leakage problems during gel casting Place the comb s into the appropriate slot s of the trays so that the sample wells are near the cathode black DNA samples will migrate toward the anode red during electrophoresis Prepare the desired concentration and amount of agarose in 1x electrophoresis buffer see Section 2 1 When the agarose solution has cooled to 50 60 C pour the molten agarose between the gates Warning Hot agarose 260 C may cause the tray to warp or craze and will decrease the lifetime of the tray Warping may also result in sample wells of uneven depth Allow 20 40 minutes for the gel to solidify at room temperature Carefully remove the comb from the solidified gel Remove the gel casting gates Submerge the gel beneath 2 to 6 mm of 1x electrophoresis buffer see Section 3 Gel and Electrophoresis Reagent Preparation Use greater depth overlay more buffer with increasing voltages to prevent pH and heat effects Removable tray UVTP gel casting using a Gel Caster or Mini Gel Caster 1 Level the Gel Caster or Mini Gel Caster using the leveling feet in the gel caster and the leveling bub ble provided 2 Disengage and slide
13. agarose to 100 ml of 1x electrophoresis buffer Table 2 1 Gel Concentration Required for DNA Separation Gel Concentration DNA Size Kb 0 50 1 30 0 75 0 8 12 1 00 0 5 10 1 25 0 4 7 1 50 0 2 3 2 5 0 01 0 5 Sieving agarose such as AmpliSize agarose Table 2 2 Gel Volume Requirements Gel Size 0 25 cm thick 0 5cmthick 0 75cmthick 1 0 cm thick Base 7x7cm 10 ml 20 ml 30 ml 40 ml 15x 7cm 20 ml 40 ml 60 ml 80 ml 15x 15cm 50 ml 100 ml 150 ml 200 ml Tray 7x7cm 10 ml 20 ml 30 ml 40 ml 7x 10 cm 15 ml 30 ml 45 ml 60 ml 15x 7cm 20 ml 40 ml 60 ml 80 ml 15x 10cm 30 ml 60 ml 90 ml 120 ml 15x 15cm 50 ml 100 ml 150 ml 200 ml 15 x 20cm 70 ml 140 ml 210 ml 280 ml 15 x 25cm 90 ml 180 ml 270 ml 360 ml 2 Add the agarose to a suitable container e g 250 ml Erlenmeyer flask Wheaton bottle etc Add the appropriate amount of 1x electrophoresis buffer see Section 3 Gel and Electrophoresis Reagent Preparation for electrophoresis buffer preparation and swirl to sus pend the agarose powder in the buffer If using an Erlenmeyer flask invert a 25 ml Erlenmeyer flask into the open end of the 250 ml Erlenmeyer flask containing the agarose The small flask acts as a reflux chamber allowing long or vigorous boiling without much evaporation Note A mark can be put on the lower flask at the same level as the liquid If evaporation occurs water can be added to bring the liquid back to the original starting level The agaros
14. be cleaned with a mild detergent and treated for 10 minutes with 3 hydrogen peroxide H O and then rinsed with 0 1 DEPC diethyl pyrocarbonate treated distilled water to eliminate RNases prior to using the Sub Cell GT systems for RNA gels 2 Consult references 1 2 for other suggestions regarding the use of DEPC in RNase decontamination Caution DEPC is a suspected carcinogen Always wear gloves eye glasses and a lab oratory coat Use caution when handling DEPC containing solutions Consult the DEPC MSDS for more information Do not attempt to RNase decontaminate Sub Cell GT parts using extreme dry heat Note Several commercial products are available for eliminating RNase contamination RNaseZAP Ambion is a safe simple and effective method that if used properly does not craze or fog the Sub Cell GT parts See manufacturer s instructions for proper use Section 5 Troubleshooting Symptoms Cause Solutions Slanted lanes bands Curved line or distortion of lanes bands Differential relative mobilities Curved bands smiles Ragged bands Band smearing and streaking Gel not fully solidified Comb warped or at an angle Bubbles in sample wells Sample spilled out of wells Unit not leveled Sample overload Sample density incorrect Sample well deformed Excessive power or heating Agarose has improper endosmosis m Salt concentration in sample too high Excessive power and heatin
15. carcinogen Always wear gloves eye glasses and a lab oratory coat Use caution when handling DEPC containing solutions Consult the DEPC MSDS Material Safety Data Sheet for more information DNA and RNA Sample Loading Dye A convenient 10x sample buffer stock consists of 50 glycerol 0 25 bromophenol blue and 0 2596 xylene cyanole FF in 1x TAE buffer Only 1 10 ml of the 10x loading dye should be prepared RNA Sample Preparation Prior to loading RNA onto an agarose formaldehyde gel prepare each RNA sample as follows 6 ul RNA in DEPC treated water 10 ul 5x MOPS buffer final concentration 1 67x 9 ul 12 3 M formaldehyde final concentration 3 7 M 25 ul formamide final concentration 50 v v Caution Formamide is a teratogen Always wear gloves eye glasses and a laboratory coat Use caution when handling formamide Consult the formamide MSDS for more information Ethidium Bromide Solution Add 10 mg of EtBr to 1 ml distilled water Bio Rad offers EtBr solutions 10 mg ml Section 4 Care and Maintenance 4 1 Cleaning Sub Cell GT Components 1 AllSub Cell GT system parts should be washed with a mild detergent solution in warm water Note Be careful not to snag or break the electrode wire in the GT base while cleaning 2 Rinse all parts thoroughly with warm water or distilled water and air dry if possible 11 4 2 Compatible Cleaning Agents Chemically compatible cleaners must be used to insure long life of parts Th
16. de Replacement The Sub Cell GT systems allow easy replacement of broken electrode wires by remov ing the banana plug electrode wire assembly and ordering a new assembly from Bio Rad Figure 4 1 Order the new assembly using the part description and catalog numbers listed in Section 6 Product Information 1 Remove the thumb screw and rubber gasket from the banana plug chamber of the GT base to release the banana plug electrode wire assembly Do not discard this thumb screw or rubber gasket keep these parts with the GT base 2 Remove the broken wire assembly by lifting upward on the banana plug Discard the bro ken assembly 3 Insert the new assembly into the banana plug chamber of the GT base Make sure the electrode wire guard guides are properly seated into the electrode wire guard slots in the bottom of the GT base 4 Replace and tighten the thumb screw and rubber gasket to secure the assembly in the base and to form a leak free seal in the banana plug holder chamber Note Test for buffer leakage by filling the base with water and checking for leakage of water through the banana plug chamber of the base If leakage occurs tighten the thumb screw Banana plug Electrode wire Banana plug Electrode wire assembly O ring Banana plug chamber Rubber gasket Thumb screw Fig 4 1 Removal of banana plug electrode wire assembly 13 4 5 RNase Decontamination Sub Cell GT parts can
17. e These rugged systems incorporate many features that make casting and running agarose gels simple and efficient The gel caster provides tape free gel casting in trays Gels can also be cast in the GT bases using specially designed wedge gates Replaceable electrode cassettes provide a simple way to replace electrode wires A comprehensive assortment of base and tray sizes including a variety of preparative analytical and multichannel pipet compatible combs makes these systems ideal for any agarose gel application Note This manual contains instructions for the Sub Cell GT electrophoresis systems only Prior to the release of the Sub Cell GT systems Bio Rad supplied similar agarose gel elec trophoresis cells the original Sub Cell DNA electrophoresis cell Wide Mini Sub cell and Mini Sub cell systems This manual does not provide information on these earlier versions Contact your local Bio Rad representative for information concerning the original Sub Cell systems Definition of Symbols A AN Caution risk of electrical shock Caution refer to accompanying documents 1 2 Safety LS AN The Sub Cell GT electrophoresis systems are designed for maximum user safety The buffer chambers are made of 3 16 inch 476 cm thick injection molded acrylic to create a leak free electrophoresis environment The safety lids surround the buffer chamber to protect the user from exposure to electrical currents All Sub Cell GT systems were designed for
18. e can be melted by boiling on a magnetic hot plate Step 4a or in a microwave oven Step 4b Caution Always wear protective gloves goggles and a lab coat while preparing and cast ing agarose gels The vessels containing hot agarose can cause severe burns if allowed to contact skin Additionally molten agarose can boil over when swirled Magnetic Hot Plate Method 4a Add a stir bar to the undissolved agarose solution Heat the solution to boiling while stirring on a magnetic hot plate Bubbles or foam should disrupt before rising to the neck of the flask Microwave Oven Method 4b Place the gel solution into the microwave Using a low to medium setting set the timer for a minimum of 5 minutes stopping the microwave oven every 30 seconds and swirling the flask gently to suspend the undissolved agarose This technique is the fastest and safest way to dissolve agarose Boil and swirl the solution until all of the small translucent agarose particles are dissolved With the small flask still in place set aside to cool to 60 C before pouring 5 2 2 Casting Agarose Gel Slabs There are several ways to cast agarose submarine gels using the Sub Cell GT systems Gels may be cast with or without a UV transparent plastic UVTP tray directly on the stage of the Sub Cell GT bases using the gel casting gates Gels may also be cast on the removable UVTP trays with the aid of the gel caster or with standard laboratory tape Casting gel
19. el to solidify at room temperature Carefully remove the comb from the solidified gel Disengage the cam peg by turning and lifting upward Slide the movable wall away from the tray Remove the tray from the Gel Caster or Mini Gel Caster Note While the gel is solidifying a light seal is formed between the gasket and the gel especially for low percentage agarose gels lt 0 8 Before moving the wall away from the tray carefully lift the tray on one side to release the seal or use a spatula to break the seal between the agarose and gasket Place the tray onto the leveled Sub Cell base so that the sample wells are near the cathode black DNA samples will migrate toward the anode red during electrophoresis Submerge the gel beneath 2 to 6 mm of 1x electrophoresis buffer see Section 3 Gel and Electrophoresis Reagent Preparation Use greater depth overlay more buffer with increasing voltages to avoid pH and heat effects Removable tray UVTP gel casting using tape 1 El ON Cum Ae Seal the ends of the UVTP gel tray securely with strips of standard laboratory tape Press the tape firmly to the edges of the gel tray to form a fluid tight seal Level the gel tray on a leveling table or workbench using the leveling bubble provided with the instrument Prepare the desired concentration and amount of agarose in 1x electrophoresis buffer see Section 2 1 When the agarose solution has cooled to 50 60 C pour the molten agarose i
20. ese include e Aqueous solutions of soaps and mild detergents Bio Rad Cleaning Concentrate catalog number 161 0722 Dishwashing liquid e Organic Solvents Hexane Aliphatic hydrocarbons Do not leave plastic parts to soak in detergents more than 30 minutes A short detergent rinse typically is all that is required Caution Do not use the following chemicals to clean Sub Cell GT parts Exposure to these chemicals may cause the plastic parts to crack craze etch or warp e Chlorinated Hydrocarbons Carbon tetrachloride Chloroform Aromatic Hydrocarbons Benzene Phenol Toluene Methyl ethyl ketone Acetone e Alcohols Methanol Ethanol Isopropyl alcohol Do not use abrasive or highly alkaline cleaners on Sub Cell GT parts Do not expose Sub Cell GT parts to temperatures gt 60 C Do not sterilize Sub Cell GT parts by autoclaving or dry heat 4 3 Maintenance Schedule Item Look For Frequency All parts Dried salts agarose Each use grease and dirt Electrical cables Breaks or fraying Each use Trays Chips or cracks Each use Electrode wires Breaks Each use Cable connections Looseness Weekly banana jacks and plugs GT base Crazing cracks Monthly or leaks Action Clean parts as described in Section 4 1 Replace cables Replace tray See Section 4 4 Electrode Cassette Replacement Replace banana jacks or banana plug holders Replace GT base or banana plug holder o ring 12 4 4 Electro
21. esent 12 Compatible Cleamng Agents ecce eimi a Nn r rE A AN PO R N E E E E NN NA ioe 12 Maintenance Schedule esses eene 12 Blectrod Replacement teile eerie ee E 13 RNase Decontamination esses eene enne enne 14 Mieri i dn 14 Product Information eee eese tees eee ee eee ee teen tatu sa tn ena tn sn sta en snae 15 Sub Cell GT Systems A 15 Sub Cel GT System ACCOSSOFIES ocean mete enviar oie ERE n 16 Related Bio Rad Products esses eerte 18 References c 21 Section 1 General Information 1 1 Introduction The Sub Cell GT instruments basic Sub Cell GT cell Wide Mini Sub cell GT and Mini Sub cell GT comprise a comprehensive and flexible gel electrophoresis system that effective ly separates nucleic acids using submerged agarose gels Submarine agarose gels are easy to cast and readily dissipate heat These gels allow sample underlaying and prevent electrical field dis continuities caused by wicks or sample well dehydration Agarose gels are ideal for the separation of DNA restriction digestions Polymerase Chain Reaction PCR amplified fragments and genomic DNA and RNA prior to Southern or northern blotting If operated correctly agarose gel submarine electrophoresis can effectively separate nucleic acids from 20 base pairs to 20 kilobase pairs in length The Sub Cell GT systems are designed for years of reproducible and rigorous us
22. f the power supply remove the safety lid and mix the running buffer as desired After the buffer has been mixed reconnect the safety lid and continue electrophoresis Table 2 3 Relative Sample Migration Rates Bromophenol Blue Cell Type Voltage Migration Rate Sub Cell GT cell 15 x 15 cm gel 75 V 3 0 cm hr Wide Mini Sub cell GT 15 x 10 cm gel 75 V 4 5 cm hr Mini Sub cell GT 7 x 10 cm gel 75 N 4 5 cm hr These sample migration rates were determined based on a 0 5 cm thick 1 096 agarose gel using Bio Rad s Molecular Biology Certified Agarose in 1x TAE electrophoresis buffer diluted from Bio Rad s Premixed 50x TAE Buffer Migration rates will vary depending on the voltage current and type of agarose or buffer used Table 2 4 DNA size migration with sample loading dyes Agarose Concentration 96 Xylene Cyanol Bromophenol Blue 0 5 1 5 4 5 Kb 400 500 bp 2 0 3 0 750 bp 100 bp 4 0 5 0 125 bp 25 bp Sieving agarose such as AmpliSize agarose 2 4 Nucleic Acid Staining and Visualization Gels can be removed from the base or gel tray for nucleic acid staining The gel can also remain on the UVTP gel tray for staining Ethidium Bromide Staining Procedure 1 Place the gel into the appropriate volume of 0 5 ug ml ethidium bromide EtBr stain for 15 30 minutes Use enough staining solution to cover the entire gel Caution Ethidium bromide is a suspected carcinogen and should be handled with extreme care Always
23. g Sample spilled out of well Incomplete digest nuclease contamination bad enzyme 14 Let gel solidify for at least 30 45 minutes Check alignment of comb Remove bubbles prior to electrophoresis Samples should have proper density Apply carefully Level unit Place on steady work bench Reduce load See sample application instructions Carefully remove comb especially from soft gels Be sure gel has solidified Cooling soft gels aids in comb removal Reduce voltage See electrophoresis instructions Consult Bio Rad about agarose Reduce salt concentration to lt 0 1 M Reduce voltage See electrophoresis instructions Apply sample carefully Increase gel thickness for large sample volumes Adjust comb height Heat sample Check enzyme activity Digest sample further Symptoms Cause Solutions Bands sharp but too few bands seen Sample wells cast through the gel Sample leaks along bottom of running surface Sample overload Too high gel percentage Incomplete digest Comb should be placed 1 to 2 mm above the base of the running surface Dilute sample Lower gel percentage Check enzyme activity digest further High MW bands Gel percentage Increase gel percentage sharp Low MW too low Switch to polyacrylamide bands smeared Gels crack Too high voltage Reduce voltage Run gel at lower gradient especially temperature with low melting temperature agarose or low
24. indoor use only Before use inspect the GT base for cracks or chips which may allow the buffer to leak from the base and cause a potential electrical hazard Additionally inspect all electrical cables banana jacks and plugs for loose connections cracks breaks or corrosion Do not use any part that is cracked charred or corroded These parts may also cause a potential electrical hazard Contact your local Bio Rad representative before using a part that may be considered hazardous During electrophoresis inspect the base and workbench for any signs of buffer leakage If leaking buffer is detected disconnect the power to the cell immediately and contact your local Bio Rad representative Power to Sub Cell GT units is supplied by an external DC voltage power supply This power supply must be ground isolated in such a way that the DC voltage output floats with respect to ground All of Bio Rad s power supplies meet this important safety requirement The recommended power supply for this apparatus is the PowerPac 300 power supply The PowerPac 300 power supply contains safety features such as no load overload rapid resis tance change and ground leak detection capabilities The maximum specified operating param eters for the Sub Cell GT systems are given in Table 1 1 Table 1 1 Sub Cell GT systems operating parameters Sub Cell Wide Mini Sub Mini Sub GT Cell Cell GT Cell GT Maximum voltage limit 200 VDC 150 VDC 150 VDC Maximum po
25. io Rad s combs are listed in Section 6 2 Loading volume is dependent upon the type of comb used i e well thickness and length and thickness of the gel 2 When loading volume is determined add standard nucleic acid sample loading dye to a final 1x concentration to make samples dense for underlaying into sample wells see Section 3 Gel and Electrophoresis Reagent Preparation for sample loading dye preparation 3 Load the samples into the wells using standard pipets Multichannel pipets can be used for loading samples only with the Bio Rad MP combs see Section 6 2 Note Sample wells are often difficult to see Well visualization can be enhanced by placing black paper or tape under the base or trays where comb placement or well formation is common 4 Place the lid on the DNA cell carefully Do not disturb the samples The Sub Cell GT system lids attach to the base in only one orientation To attach the lid correctly match the red and black banana jacks on the lid with the red and black banana plugs of the base 5 Power requirements vary depending on gel thickness length and concentration and type of electrophoresis buffer used Refer to Tables 2 3 and 2 4 for relative sample migration rates and for DNA size migration with sample loading dyes for the different Sub Cell GT systems Note Buffer recirculation is not required for most standard DNA and RNA agarose gel electrophoresis If buffer recirculation is required simply turn of
26. nto the gel tray Warning Hot agarose 260 C may cause the tray to warp or craze and will decrease the lifetime of the tray Warping may also result in sample wells of uneven depth Allow 20 40 minutes for the gel to solidify at room temperature Carefully remove the comb from the solidified gel Remove the tape from the edges of the gel tray Place the tray onto the leveled Sub Cell base so that the sample wells are near the cath ode black DNA samples will migrate toward the anode red during electrophoresis Submerge the gel under 2 to 6 mm buffer see Section 3 Gel and Electrophoresis Reagent Preparation Use greater depth overlay more buffer with increasing voltages to avoid pH and heat effects 2 3 Electrophoresis After the agarose gel has solidified sample loading and electrophoresis can begin Agarose gels can be run in many different types of electrophoresis buffers Nucleic acid agarose gel electrophoresis is usually conducted with either Tris Acetate EDTA TAE buffer or Tris Borate EDTA TBE buffer While TAE buffer provides faster electrophoretic migration of linear DNA and better resolution of supercoiled DNA TBE buffers have a stronger buffering capacity for longer or higher voltage electrophoresis runs Bio Rad offers premixed 50x TAE and 10x TBE buffers as well as individual buffer reagents for use with the Sub Cell GT systems KL Prepare samples for gel loading The maximum sample loading volumes for B
27. r Number mm mm Capacity ul 170 4342 1 1 50 43 43 325 7 170 4333 8 1 50 5 54 41 6 170 4332 15 1 50 2 59 19 4 Well volume capacity was determined based on a well depth of 0 5 cm 6 3 Related Bio Rad Products Power Supplies 165 5050 PowerPac 300 Power Supply 100 120 V 165 5051 PowerPac 300 Power Supply 220 240 V Blotting Membranes 161 0153 Zeta Probe Positively Charged Nylon Blotting Membrane sheets 9 x 12 cm 15 161 0154 Zeta Probe Positively Charged Nylon Blotting Membrane sheets 10 x 15 cm 15 161 0155 Zeta Probe Positively Charged Nylon Blotting Membrane sheets 15 x 15 cm 15 161 0156 Zeta Probe Positively Charged Nylon Blotting Membrane sheets 15 x 20 cm 15 161 0157 Zeta Probe Positively Charged Nylon Blotting Membrane sheets 20 x 20 cm 15 161 0158 Zeta Probe Positively Charged Nylon Blotting Membrane sheets 20 x 25 cm 3 161 0159 Zeta Probe Positively Charged Nylon Blotting Membrane roll 30 cm x 3 3 m 1 161 0165 Zeta Probe Positively Charged Nylon Blotting Membrane roll 20 cm x 3 3 m 1 161 0190 Zeta Probe GT Genomic Tested Positively Charged Nylon Blotting Membrane sheets 9 x 12 cm 15 161 0191 Zeta Probe GT Genomic Tested Positively Charged Nylon Blotting Membrane sheets 10 x 15 cm 15 18 161 0192 161 0193 161 0194 161 0195 161 0196 161 0197 161 0090 161 0091 161 0092 161 0093 161 0094 161 0095 161 0096
28. s on the base stages l 2 3 Level the Sub Cell base using the leveling bubble provided Slide the gel casting gates into the slots at opposite ends of the gel stage Place the comb s into the appropriate slot s of the base so that the sample wells are near the cathode black DNA samples will migrate toward the anode red during electrophoresis Prepare the desired concentration and amount of agarose in 1x electrophoresis buffer see Section 2 1 When the agarose solution has cooled to 50 60 C pour the molten agarose between the gates Warning Hot agarose gt 60 C may cause the plastic in the cell to warp or craze and will decrease the lifetime of the Sub Cell base Warping may also result in sample wells of uneven depth Allow 20 40 minutes for the gel to solidify at room temperature Carefully remove the comb from the solidified gel Remove the gel casting gates Submerge the gel beneath 2 to 6 mm of 1x electrophoresis buffer see Section 3 Gel and Electrophoresis Reagent Preparation Use greater depth overlay more buffer with increasing voltages to avoid pH and heat effects Casting Gels on the Base Stage With UVTP Tray 1 2 Level the cell using the leveling bubble provided Place the UVTP tray on the cell base stage Note The Mini Sub cell GT requires the 7 x 7 cm UVTP tray for casting in the base The Wide Mini Sub cell GT requires the 15 x 7 cm UVTP tray and the Sub Cell GT sys tem requires the 15 x
29. tem or with CCD based digitized image analysis systems e g Gel Doc 1000 UV fluorescent gel documentation system Gels are generally pho tographed with a yellow orange or red interference filter Red filters generally give the cleanest background Bio Rad offers a full line of standard photography and CCD based imaging systems for nucleic acid gel analysis 2 5 Note on Blotting Nucleic acids within the gel can be transferred to membranes using the techniques of Southern and Northern blotting It is beyond the scope of this instruction manual to include blotting procedures Consult references and 2 for blotting techniques Bio Rad offers a full line of nitrocellulose and positively charged nylon membranes as well as vacuum and elec trophoretic blotting apparatus for Southern and Northern blotting Section 3 Gel and Electrophoresis Reagent Preparation RNA Agarose Formaldehyde Gels For 100 ml of a 1 agarose formaldehyde gel prepare as follows 62 ml of 1 6 melted agarose 20 ml 5x MOPS electrophoresis buffer 1x final concentration 18 ml 12 3 M 37 5 formaldehyde 2 2 M final concentration Caution Formaldehyde solutions and formaldehyde vapors are toxic When handling solu tions or gels that contain formaldehyde use a chemical hood Always wear gloves eye glass es and a laboratory coat while using formaldehyde See the MSDS for safety information Nucleic Acid Electrophoresis Buffers 2 DNA agarose gel electrophoresis
30. tem Components Each of the Sub Cell GT systems comes with the components listed in Table 1 2 see Figure 1 1 for part description Check your instrument 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 2 Sub Cell GT System Components Sub Cell GT Wide Mini Sub Cell Mini Sub Cell System GT System GT System Item Quantity Quantity Quantity GT Base buffer chamber 1 1 1 Gel Casting Gates 2 2 2 Safety Lid and Cables 1 1 1 UVTP Gel Tray 1 1 1 Fixed Position Comb 2 2 2 15 well 1 5 mm thick 15 well 1 5 mm thick 8 well 1 5 mm thick 20 well 1 5 mm thick 20 well 1 5 mm thick 15 well 1 5 mm thick Leveling Bubble 1 1 1 Gel Caster optional 1 1 1 Instruction Manual 1 1 1 Electrical cables Fixed height comb Comb slots a ee Banana plug Electrode wire assembly Electrical leads Gel casting gates GT Base Safety Lid removal peg Leveling feet Fixed height comb Leveling feet UVTP gel tray Cam lever Gel caster Leveling bubble uu Leveling feet Fig 1 1 Sub Cell GT parts 1 4 Specifications Sub Cell GT Wide Mini Sub Cell Mini Sub Cell System GT System GT System GT base footprint LxWxH 42x19 5x10cm 26x20x7 5cm 26x12x6 5cm GT base buffer volumet 1 500 2 000 ml 650 900 ml 265 320 ml GT base gel si
31. the movable wall to the open end of the Gel Caster or Mini Gel Caster by turning and lifting the cam peg upward Note If casting more than one gel with the Gel Caster add the removable gel casting wall to the gel caster The removable wall will allow casting of two 15 x 10 cm trays four 7 x10 cm trays or one 15 x 10 cm and one 15 x15 cm trays 3 Place the open edge of the UVTP tray against the fixed wall of the Gel Caster or Mini Gel Caster 4 Slide the movable wall against the edge of the UVTP tray Figure 2 1 5 Tosealthe open tray ends engage the cam peg by turning and pressing downward simul taneously 6 When the cam peg has dropped into the appropriate slot turn the peg in either direction until resistance is felt This action seals the edges of the tray for casting 7 Place the comb s into the appropriate slot s of the tray Lift cam lever up Movable wall of gel caster Fixed wall of gel caster Engage and seal press down and rotate Fig 2 1 Sealing the UVTP tray for gel casting 8 Prepare the desired concentration and amount of agarose in 1x electrophoresis buffer see Section 2 1 When the agarose solution has cooled to 50 60 C pour the molten agarose between the gates Warning Hot agarose gt 60 C may cause the tray to warp or craze and will decrease the lifetime of the tray Warping may also result in sample wells of uneven depth 10 11 12 13 Allow 20 40 minutes for the g
32. wear gloves eye glasses and a laboratory coat Dispose of used EtBr solutions and gels appropriately Review EtBr Material Safety Data Sheet MSDS for proper disposal methods 2 Destain the gel for 10 30 minutes in dH O using the same volume used for staining Note Ethidium Bromide can be removed from the DNA with extended destaining This will cause lower sensitivity of detection However insufficient destaining will create higher background fluorescence 3 Rinse the gel briefly with dH O to remove any residual staining solution 4 Place the gel on a UV transilluminator for nucleic acid visualization and analysis DNA Ethidium Bromide complexes may be illuminated with UV light of 254 302 or 366 nm Sensitivity decreases with illumination at higher wavelengths However nicking of DNA will increase below 302 nm Table 2 5 gives the percentage of transmittance of UV light through 1 4 64 cm UV transparent plastic Note Nucleic acids in the gel can be visualized through the UVTP trays If a UVTP tray is not used place household plastic wrap between the UV transilluminator and the gel to avoid contaminating the transilluminator with nucleic acids or EtBr Table 2 5 Percent UV Transmittance through 1 4 64 cm UV Transparent Plastic UVTP Approximate Wavelength nm Transmittance 254 0 302 80 360 90 5 Photograph the gel using standard cameras and film e g Bio Rad s Standard Polaroid Gel Documentation Sys
33. wer limit 40 Watts 45 Watts 10 Watts Maximum Buffer temperature 40 C 40 C 40 C Current to the cell provided from the external power supply enters the unit through the lid assembly providing a safety interlock Current to the cell is broken when the lid is removed Do not attempt to circumvent this safety interlock and always turn the power supply off before removing the lid or when working with the cell Important These Bio Rad instruments are certified to meet IEC 1010 1 safety stan dards IEC certified products are safe to use when operated in accordance with the instruction manual This instrument should not be modified in any way Alteration of this instrument will e Void the manufacturer s warranty e Void the IEC 1010 1 safety certification e Create a potential safety hazard IEC 1010 1 certification applies to equipment designed to be safe between the operating temperatures of 4 C and 40 C and altitudes up to 2 000 meters Instruments are also safe at a max imum relative humidity of 80 for temperatures up to 31 C decreasing linearly to 50 96 at 40 C Bio Rad is not responsible for any injury or damage caused by the use of this instru ment for purposes other than those for which it is intended or by modifications of the instrument not performed by Bio Rad or an authorized agent No user serviceable parts are contained in this apparatus To insure electrical safety do not attempt to service this apparatus 1 3 Sys
34. ze 15 x 15cm 15x 7 cm 7x cm Gel tray sizes 15 x 10 cm 15x 7 cm 7xcm 15x15cm 15x 10 cm 7x10cm 15 x 20cm 15 x 25cm Construction GT base Molded clear plastic Gel casting gates Aluminum Safety lid Molded clear plastic Banana plug electrode cassette Molded polycarbonate Banana plugs Gold plated brass 4 4 cm length Electrodes Platinum 0 25 mm diameter Electrical cables Dual 20 AWG tinned copper wire cable Flame retardant polyurethane insulation jacket Electrical leads Nickel silver Gel tray UV transparent acrylic plastic UVTP Combs Molded plastic and machined acrylic Gel casting device Polycarbonate 0 64 cm silicon foam t GT base buffer volumes will vary depending on the size and thickness of the gel used Section 2 Operating Instructions Note See Section 3 Gel and Electrophoresis Reagent Preparation for information on the preparation of RNA gels See References 1 and 2 for more information on DNA and RNA electrophoresis 2 1 DNA Gel Preparation DNA agarose gels can be used to separate and visualize DNA of various sizes Before cast ing an agarose gel consult Table 2 1 to determine the appropriate percent agarose gel to use based on the size of DNA to be separated Procedure 1 Determine the amount of agarose grams required to make the desired agarose gel con centration and volume Use Tables 2 1 and 2 2 as a guide for agarose concentration and gel volume requirements Example For a 1 agarose gel add 1 gram of
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