Sub-Cell® GT Agarose Gel Electrophoresis Systems - Bio-Rad
Contents
1. Disengage and slide 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 using two 15 x 10 cm trays four 7 x10 cm trays or one 15 x 10 cm and one 15 x15 cm trays Place the open edge of the UVTP tray against the fixed wall of the Gel Caster or Mini Gel Caster Slide the movable wall against the edge of the UVTP tray Figure 2 1 Lift cam lever up Engage and seal Movable wall press down and rotate of gel caster Fixed wall of gel caster Fig 2 1 Sealing the UVTP tray for gel casting 9 10 11 To seal the open tray ends engage the cam peg by turning and pressing downward simultaneously 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 Place the comb s into the appropriate slot s of the tray 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 Allow 20 40 min for the ge2. 165 5034 GS Gene Linker UV Chamber 100 VAC 18 Catalog Number Product Description Gel Reagents and Standards 161 3101 Certified Molecular Biology Agarose 125 g 161 3104 Certified PCR Agarose 125 g 161 3107 Certified Low Range Ultra Agarose 125 g 170 8200 AmpliSize DNA Molecular Ruler 50 2 000 bp 250 ul 161 0404 Bromophenol Blue 10 g 161 0423 Xylene Cyanole FF 25 g 161 0433 Ethidium Bromide Solution 10 ml 10 mg ml Electrophoresis Buffers 161 0733 10x Tris Boric Acid EDTA TBE 1 161 0743 50x Tris Acetic Acid EDTA TAE 1 161 0719 Tris 1 kg DNA Gel Image Analysis and Documentation Systems 170 8190 Molecular Imager Gel Doc XR System PC and MAC 170 8251 Molecular Imager ChemiDoc XRS System PC 170 8252 Molecular Imager ChemiDoc XRS System MAC Section 7 References 1 Sambrook Fritsch and Maniatis Molecular Cloning A Laboratory Manual Second Edition Cold Spring Harbor Laboratory Press 1989 2 Current Protocols in Molecular Biology Greene Publishing Associates and Wiley Interscience 1989 Additional Reading 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
3. Winberg G and Hammarskjold M L Nucleic Acids Res 8 253 1980 OM NDA AR amp 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 IEC 1010 1 is an internationally accepted electrical safety standard for laboratory instruments 19 20 21 Life Science Group 10016027 RevC US EG Bio Rad Laboratories Inc Web site www bio rad com USA 800 424 6723 Australia 61 2 9914 2800 Austria 01 877 89 01 Belgium 09 385 55 11 Brazil 55 31 3689 6600 Canada 905 364 3435 China 86 20 8732 2339 Czech Republic 420 241 430 532 Denmark 44 52 10 00 Finland 09 804 22 00 France 01 47 95 69 65 Germany 089 31 884 0 Greece 30 210 777 4396 Hong Kong 852 2789 3300 Hungary 36 1 459 6100 India 91 124 4029300 Israel 03 963 6050 Italy 39 02 216091 Japan 03 6361 7000 Korea 82 2 3473 4460 Mexico 52 555 488 7670 The Netherlands 0318 540666 New Zealand 0508 805 500 Norway 23 38 41 30 Poland 48 22 331 99 99 Portugal 351 21 472 7700 Russia 7 495 721 14 04 Singapore 65 6415 3188 South Africa 27 861 246 723 Spain 34 91 590 5200 Sweden 08 555 12700 Switzerland 061 717 95 55 Taiwan 886 2 2578 7189 United Kingdom 020 8328 2000 10 0082 0100
4. 2 Compatible Cleaning Agents Chemically compatible cleaners must be used to insure long life of parts These 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 Chlorinated hydrocarbons Carbon tetrachloride Chloroform Aromatic hydrocarbons Benzene Phenol Toluene Methyl ethyl ketone Acetone 11 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 Action All parts Dried salts agarose Each use Clean parts as described in grease and dirt Section 4 1 Electrical cables Breaks or fraying Each use Replace cables Trays Chips or cracks Each use Replace tray Electrode wires Breaks Each use See Section 4 4 Electrode assembly replacement Cable connections Looseness Weekly Replace banana jacks or banana jacks and plugs banana plug holders GT base Crazing cracks M
5. 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 with 15 x 7 cm tray 170 4485 Wide Mini Sub Cell GT System with 15 x 7 cm tray and mini gel caster 170 4406 Mini Sub Cell GT System with 7 x 7 cm tray 170 4486 Mini Sub Cell GT System with 7 x 7 cm tray and mini gel caster Sub Cell Systems with PowerPac Basic Power Supply 100 120 220 240 V 164 0302 Sub Cell GT Cell and PowerPac Basic Power Supply 164 0301 Wide Mini Sub Cell GT Cell and PowerPac Basic Power Supply 164 0300 Mini Sub Cell GT Cell and PowerPac Basic Power Supply 14 6 2 Sub Cell GT System Accessories Catalog Number Product Description Sub Cell GT Systems 170 4390 170 4391 170 4412 170 4392 170 4393 170 4415 170 4416 170 4417 170 4418 170 4419 Sub Cell GT Base Sub Cell GT Safety Lid with Cables Gel Caster Full size Sub Cell GT QuickSnap Electrode Assembly Anode red Sub Cell GT QuickSnap Electrode Assembly Cathode black Sub Cell GT Gel Casting Gates 2 Sub Cell GT UVTP Gel Tray 15 x 10 cm Sub Cell GT UVTP Gel Tray 15 x 15 cm Sub Cell GT UVTP Gel Tray 15 x 20 cm Sub Cell GT UVTP Gel Tray 15 x 25 cm Wide Mini Sub Cell GT Systems 170 4370 170 4371 170 4422 170 4372 170 4373 170 4425 170 4416 170 4426 Wide Mini Sub Cell GT Base Wide Mini Sub Cell
6. GT Safety Lid with Cables Mini Gel Caster Wide Mini Sub Cell GT QuickSnap Electrode Assembly Anode red Wide Mini Sub Cell GT QuickSnap Electrode Assembly Cathode black Wide Mini Sub Cell GT Gel Casting Gates 2 Sub Cell GT UVTP Wide Mini Gel Tray 15 x 10 cm Sub Cell GT UVTP Wide Mini Gel Tray 15 x 7 cm Mini Sub Cell GT Systems 170 4360 170 4361 170 4422 170 4362 170 4363 170 4434 170 4435 170 4436 Mini Sub Cell GT Base Mini Sub Cell GT Safety Lid with Cables Mini Gel Caster Mini Sub Cell GT QuickSnap Electrode Assembly Anode red Mini Sub Cell GT QuickSnap Electrode Assembly Cathode black Mini Sub Cell GT Gel Casting Gates 2 Sub Cell GT UVTP Mini Gel Tray 7 x 10 cm Sub Cell GT UVTP Mini Gel Tray 7 x 7 cm 15 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 4449 30 1 50 2 69 20 2 170 4447 20 0 75 4 84 18 2 170 4448 20 1 50 4 84 36 3 170 4445 15 0 75 5 52 20 7 170 4446 15 1 56 5 52 41 4 170 4443 10 0 75 9 87 37 0 170 4444 10 1 50 9 87 74 0 Adjustable Height Combs For Sub Cell GT and Wide Mini Sub Cell GT Systems Adjustable height combs require a comb holder catalog number 170 4320 Catalog Well Thickness Well Width Well Volume Number Number mm mm Capacity ul 170 4344 30 1 50 2 69 20 2 170 4321 20 0 75 4 84 18 2 170 4322 20 1 50 4 84 36 4 170 4323
7. Sig 1109
8. conform to IEC 1010 1 standards safe between the operating temperatures of 4 C and 40 C and altitudes up to 2 000 meters safe at a maximum relative humidity of 80 for temperatures up to 31 C and for temperatures higher than 31 C safe at a maximum humidity that decreases linearly to 50 at 40 C 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 of the instrument not performed by Bio Rad or an authorized agent There are no user serviceable parts in this apparatus To insure electrical safety do not attempt to service this apparatus 1 3 System Components Each of the Sub Cell GT systems comes with the components listed in Table 1 2 see Figure 1 1 page 3 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 dl 1 Gel Casting Gates optional 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 C
9. sample loading volumes for Bio Rad s combs are listed in Section 6 2 Loading volume is dependent upon the type of comb used 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 only be used for loading samples 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 in the are of comb placement and well formation 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 agarose concentration and type of electrophoresis buffer used Refer to Tables 2 3 and 2 4 below for relative sample migration rates for the different Sub Cell GT systems and for DNA size migration with sample loading dyes Note Buffer recirculation is not required for most standard DNA and RNA agarose gel electrophoresis If buffer
10. the gel tray 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 4 Allow 20 40 min for the gel to solidify at room temperature 5 Carefully remove the comb from the solidified gel 6 Remove the tape from the edges of the gel tray 7 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 8 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 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 buffers provide 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 1 Prepare samples for gel loading The maximum
11. 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 5 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 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 a UV transparent plastic UVTP tray directly on the gel 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 gels on the base stage with the UVTP tray 1 2 Level the cell using the leveling bubble provided Place the UVTP tray on the gel stage Note The Mini Sub Cell GT requires the 7 x 7 cm UVTP tray for casting in the GT base The Wide Mini Sub Cell GT requires the 15 x 7 cm UVTP tray and the Sub Cell GT system requires the 15 x 15 cm UVTP tray for casting in the GT base Slide the gel casting gates into the slots at opposite ends of the GT gel stage Insure that 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
12. 15 0 75 5 52 20 7 170 4324 15 1 50 5 02 41 4 170 4325 10 0 75 9 87 37 0 170 4326 10 1 50 9 87 74 0 Preparative Combs For Sub Cell GT and Wide Mini Sub GT Systems Adjustable height combs require a comb holder catalog number 170 4320 Catalog Well Thickness Well Width Well Volume Number Number mm mm Capacity ul 170 4442 4 1 50 26 42 200 0 170 4441 2 1 50 50 29 377 0 170 4440 1 1 50 106 43 800 0 170 4328 1 3 00 106 43 1 596 0 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 4456 26 0 75 2 91 10 9 170 4457 26 1 50 2 91 21 8 170 4454 18 0 75 2 91 10 9 170 4455 18 1 50 2 91 21 8 170 4452 14 0 75 5 82 21 8 170 4453 14 1 50 5 82 43 6 170 4450 10 0 75 5 82 21 8 170 4451 10 1 80 5 82 43 6 16 Fixed Height Combs for Mini Sub Cell GT Catalog Well Thickness Well Width Well Volume Number Number mm mm Capacity ul 170 4464 15 0 75 2 59 9 7 170 4465 15 1 50 2 59 19 4 170 4462 8 0 75 5 54 20 8 170 4463 8 1 50 5 54 41 6 170 4461 2 1 50 20 32 152 4 170 4460 p 1 50 43 43 325 7 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 Number Number mm mm Capacity ul 170 4332 15 1 00 2 59 12 95 170 4333 8 1 00 5 54 27 7 170 4342 1 3 00 43 4
13. 15x 15cm 50m 100 m 150 m 200 m 15x 20cm 70m 140m 210m 280 m 15x25cm 90m 180m 270m 360 m 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 suspend 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 agarose can be melted by boiling on a magnetic hot plate Step 4a or in a microwave oven Step 4b Caution Always wear protective gloves safety glasses and a lab coat while preparing and casting 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
14. 3 651 45 Well volume capacity was determined based on a well depth of 0 5 cm 6 3 Related Bio Rad Products Power Supplies 164 5050 PowerPac Basic Power Supply 100 120 220 240 V 164 5052 PowerPac HC Power Supply 100 120 220 240 V Blotting Membranes 162 0153 Zeta Probe Positively Charged Nylon Blotting Membrane sheets 9 x 12 cm 15 162 0154 Zeta Probe Positively Charged Nylon Blotting Membrane sheets 10 x 15 cm 15 162 0155 Zeta Probe Positively Charged Nylon Blotting Membrane sheets 15 x 15 cm 15 162 0156 Zeta Probe Positively Charged Nylon Blotting Membrane sheets 15 x 20 cm 15 162 0157 Zeta Probe Positively Charged Nylon Blotting Membrane sheets 20 x 20 cm 15 162 0158 Zeta Probe Positively Charged Nylon Blotting Membrane sheets 20 x 25 cm 3 162 0159 Zeta Probe Positively Charged Nylon Blotting Membrane roll 30 cm x 3 3 m 1 162 0165 Zeta Probe Positively Charged Nylon Blotting Membrane roll 20 cm x 3 3 m 1 162 0190 Zeta Probe GT Genomic Tested Positively Charged Nylon Blotting Membrane sheets 9 x 12 cm 15 162 0191 Zeta Probe GT Genomic Tested Positively Charged Nylon Blotting Membrane sheets 10 x 15 cm 15 17 Catalog Number Product Description 162 0192 Zeta Probe GT Genomic Tested Positively Charged Nylon Blotting Membrane sheets 15 x 15 cm 15 162 0193 Zeta Probe GT Genomic Tested Positively Charged Nylon Blotting Membrane sheets 15 x 20 cm 15 162 0194 Zeta Prob
15. 80 366 90 Photograph the gel using standard cameras and film e g Bio Rad s Standard Polaroid Gel Documentation System or with CCD based digitized image analysis systems e g Gel Doc 1000 UV fluorescent gel documentation system Gels are generally photographed 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 1 and 2 for blotting techniques Bio Rad offers a full line of nitrocellulose and positively charged nylon membranes as well as vacuum and electrophoretic 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 solutions or gels that contain formaldehyde use a chemical hood Always wear gloves safety glasses and a laboratory coat when using formalde
16. Sub Cell GT Agarose Gel Electrophoresis Systems Instruction Manual Catalog 170 4401 to 170 4406 170 4481 to 170 4486 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 1 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 GT system components and accessories be inspected for damage cleaned as recommended in this manual and rinsed thoroughly
17. afety glasses and a laboratory coat Use caution when handling DEPC containing solutions Consult the DEPC MSDS for more information Do not attempt to decontaminate RNase from Sub Cell GT parts using extreme dry heat Note Several commercial products are available for eliminating RNase contamination RNaseZAP Ambion is asafe 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 mr Salt concentration in sample too high Excessive power and heating Sample spilled out of well Incomplete digestion nuclease contamination bad enzyme Let gel solidify for at least 30 45 min 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 s
18. anual 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 A Caution risk of electrical shock Caution refer to accompanying documents 1 2 Safety A A The Sub Cell GT electrophoresis systems are designed for maximum user safety The buffer chambers are made of 2 75 mm 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 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 t
19. aster optional 1 1 i Instruction Manual 1 A 1 Safety lid Electrical cables Electrical leads Fluorescent ruler Banana plug electrode wire assembly UV transparent gel tray Gel casting gates Mini Sub Cell GT Base Safety lid removal tab Gel stage Fixed height ome GQ Fluorescent ruler UV transparent gel tray Cam lever Gel caster Leveling bubble Leveling feet Fig 1 1 Mini Sub Cell GT components 1 4 Specifications Sub Cell GT Wide Mini Sub Mini Sub Cell System Cell GT System GT System GT base footprint L x W x H 40 5 x 18 x 9 4 cm 25 5x17 8x6 8cm 25 5x9 2x5 6cm GT base buffer volumet 1 500 2 000 ml 650 900 ml 265 320 ml GT base gel size 15x 15cm 15x7 cm 7x7cm Gel tray sizes 15x 10cm 15x7 cm 7x7cm 15x 15cm 15x10cm 7x10cm 15 x 20 cm 15x 25 cm Construction GT base Gel casting gates Safety lid Electrode wire guard Banana plugs Electrodes Electrical cables Electrical leads Gel tray Combs Gel casting device Molded clear plastic Aluminum Molded clear plastic Molded polycarbonate Gold plated brass 4 4 cm length Platinum 0 25 mm diameter Dual 20 AWG tinned copper wire cable Flame retardant polyurethane insulation jacket Nickel silver UV transparent acrylic plastic UVTP Molded plastic and machined acrylic Polycarbonate 0 64 cm silicon foam t GT base buffer volumes will vary depending on the size and thickness of the
20. e GT Genomic Tested Positively Charged Nylon Blotting Membrane sheets 20 x 20 cm 15 162 0195 Zeta Probe GT Genomic Tested Positively Charged Nylon Blotting Membrane sheets 20 x 25 cm 3 162 0196 Zeta Probe GT Genomic Tested Positively Charged Nylon Blotting Membrane roll 30 cm x 3 3 m 1 162 0197 Zeta Probe GT Genomic Tested Positively Charged Nylon Blotting Membrane roll 20 cm x 3 3 m 1 162 0090 Supported Nitrocellulose Membrane 0 45 micron sheets 7 x 8 4 cm 10 162 0091 Supported Nitrocellulose Membrane 0 45 micron sheets 10 x 15 cm 10 162 0092 Supported Nitrocellulose Membrane 0 45 micron sheets 15 x 15 cm 10 162 0093 Supported Nitrocellulose Membrane 0 45 micron sheets 20 x 20 cm 10 162 0094 Supported Nitrocellulose Membrane 0 45 micron roll 30 cm x 3 m 1 162 0095 Supported Nitrocellulose Membrane 0 20 micron sheets 7 x 8 4 cm 10 162 0096 Supported Nitrocellulose Membrane 0 20 micron sheets 15 x 15 cm 10 162 0097 Supported Nitrocellulose Membrane 0 20 micron roll 30 cm x 3 m 1 Vacuum Blotting Apparatus 165 5000 Model 785 Vacuum Blotter 165 5001 Model 785 Vacuum Blotter System 120 VAC 165 5002 Model 785 Vacuum Blotter System 220 240 VAC Semi Dry Transfer Cells 170 3940 Trans Blot SD Semi Dry Electrophoresis Transfer Cell UV Crosslinking Chamber 165 5031 GS Gene Linker UV Chamber 120 VAC 165 5032 GS Gene Linker UV Chamber 220 VAC 165 5033 GS Gene Linker UV Chamber 240 VAC
21. 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 casting an agarose gel consult Table 2 1 page 5 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 concentration and volume Use Tables 2 1 and 2 2 page 5 as a guide for agarose concentration and gel volume requirements Example For a 1 agarose gel add 1 gram of agarose to 100 ml of 1x electrophoresis buffer Table 2 1 Gel concentration required for DNA separation 2 Gel Concentration DNA Size 0 50 1 30 Kb 0 75 800 bp 10 Kb 1 00 500 bp 10 Kb 1 25 400 bp 7 Kb 1 50 200 bp 3 Kb 2 00 100 bp 2 5 Kb 3 00 40 bp 2 Kb 4 00 10 400 bp Sieving agarose such as Certified PCR agarose Sieving agarose such as Certified low range ultra agarose Table 2 2 Gel volume requirements Gel Size thickness 0 25 cm 0 5 cm 0 75 cm 1 0 cm Base 7x7cm 10m 20m 30m 40 m 15x7cm 20m 40 m 60m 80 mi 15x 15cm 50m 100m 150m 200 m Tray 7x7cm 10m 20m 30m 40 mi 7x10cm 15m 30m 45m 60 m 15x7 cm 20 ml 40m 60m 80 mi 15x 10cm 30m 60m 90m 120m
22. hat 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 these units is the PowerPac Basic power supply The PowerPac Basic power supply contains safety features such as no load overload rapid resistance change and ground leak detection capabilities The maximum specified operating parameters 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 power limit 40 Watts 45 Watts 10 Watts Maximum Buffer temperature 40 C 40 C 40 C Electric current to the cell from the external power supply enters the unit through the lid assembly which provides 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 standards 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 The Sub Cell GT systems
23. hyde See the MSDS for safety information Nucleic acid electrophoresis buffers DNA agarose gel electrophoresis is usually performed using either Tris Acetate EDTA TAE or Tris Borate EDTA TBE While TAE buffers provide 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 for use with the Sub Cell GT systems RNA formaldehyde gels require a MOPS 3 N morpholino propanesulfonic acid electrophoresis buffer 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 Borate EDTA TBE 89 mM tris pH 7 6 89 mM boric acid 2 mM 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 F
24. ic 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 discontinuities 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 use 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 casting 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 electrophoresis cells the original Sub Cell DNA electrophoresis cell Wide Mini Sub cell and Mini Sub cell systems This m
25. ill to a final volume of 1 liter with DEPC treated distilled water Caution DEPC is a suspected carcinogen Always wear gloves safety glasses and a laboratory coat Use caution when handling DEPC containing solutions Consult the DEPC MSDS Material Safety Data Sheet for more information 10 DNA and RNA sample loading dye 2 A convenient 10x sample buffer stock consists of 50 glycerol 0 25 bromophenol blue and 0 25 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 safety 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 m Section 4 Care and Maintenance 4 1 Cleaning Sub Cell GT Components 1 All Sub 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 4
26. l 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 12 13 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 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 into
27. ng solution to cover the entire gel Caution Ethidium bromide is a suspected carcinogen and should be handled with extreme care Always wear gloves safety glasses and a laboratory coat Dispose of used EtBr solutions and gels appropriately Review EtBr Material Safety Data Sheet MSDS for proper disposal methods Destain the gel for 10 30 min in dH O with 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 Rinse the gel briefly with dH O to remove any residual staining solution 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
28. olidified 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 hickness for large sample volumes Adjust comb height Heat sample Check enzyme activity Digest sample further 13 Symptoms Cause Solutions Sample wells cast Comb should be placed 1 to 2 mm through the gel Sample above the base of the running surface Bands sharp but too few bands seen High MW bands sharp Low MW bands smeared leaks along bottom of running surface Sample overload Dilute sample Gel agarose percentage too high Lower agarose percentage Incomplete digestion Check enzyme activity digest further Gel agarose percentage too low Increase agarose percentage Switch to polyacrylamide Gels crack Too high voltage gradient Reduce voltage Run gel at lower especially with low melting temperature temperature agarose or low 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
29. onthly Replace GT base 4 4 Electrode Replacement The Sub Cell GT systems allow easy replacement of broken electrode wires by removing the banana plug electrode wire assembly and ordering a new electrode 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 broken wire assembly by placing one finger on the banana plug and by pressing with another finger the QuickSnap feature on the outside of the upper part of the electrode wire assembly and lifting upward Discard the broken assembly 2 Insert the new assembly into the electrode assembly chamber of the GT base Make sure the tab of the QuickSnap is properly seated in the slot of the electrode assembly chamber Electrode wire Banana plug Banana plug Electrode wire a assembly O ity Electrode s MJ assembly Pan Za chamber y a Fig 4 1 Removal of banana plug electrode wire assembly 12 4 5 RNase Decontamination Sub Cell GT parts can 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 1 2 Consult references 1 and 2 for other suggestions regarding the use of DEPC in RNase decontamination Caution DEPC is a suspected carcinogen Always wear gloves s
30. problems during gel casting Note If leakage occurs while pouring the gel on the casting tray atop the stage chill the casting gates in the freezer for 2 3 minutes Place the casting gates into the slots when ready to pour the gel The chilled casting gates will prevent the gel solution from leaking out of the tray and into the chambers 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 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 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 bubble provided
31. recirculation is required simply turn off 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 with electrophoresis Table 2 3 Relative sample migration rates Bromophenol Blue Cell Type Voltage Migration Rate Sub Cell GT cell 15 x 15 cm gel 75V 3 0 cm hr Wide Mini Sub cell GT 15 x 10 cm gel 75V 4 5 cm hr Mini Sub cell GT 7 x 10 cm gel 75V 4 5 cm hr These sample migration rates were determined based on a 0 5 cm thick 1 0 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 Xylene Cyanol Bromophenol Blue 0 5 1 5 4 5 Kb 400 500 bp 2 0 3 0 750 bp 100 bp gt 3 0 125 bp 25 bp Sieving agarose such as Certified PCR agarose Sieving agarose such as Certified low range ultra agarose 2 4 Nucleic Acid Staining and Visualization Gels can be removed from the Sub Cell GT 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 min Use enough staini
32. 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 23 2 4 25 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 General Information Introduction Safety System Components Specifications Operating Instructions DNA Gel Preparation Casting Agarose Gel Slabs Electrophoresis Nucleic Acid Staining and Visualization Note on Blotting Gel and Electrophoresis Reagent Preparation Care and Maintenance Cleaning Sub Cell GT Components Compatible Cleaning Agents Maintenance Schedule Electrode Replacement RNase Decontamination Troubleshooting Product Information Sub Cell GT Systems Sub Cell GT System Accessories Related Bio Rad Products References Page oo oor A HRN _ o i h a a ae ONnNNnN b A i i mb NORA 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 effectively separates nucle
Download Pdf Manuals
Related Search