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C.B.S. SCIENTIFIC DGGE-1001, Instruction Manual

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C.B.S. SCIENTIFIC DGGE-1001 Instruction Manual Download Page 21

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858-755-4959 

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3.7   Running the Gels 

 

1.  Load samples at 1:1 with neutral dye.  Load 5-10 ug Genomic DNA/well or 1-2 ug 

cloned (B-globin)/well.  Determine concentration by O.D. 260. 

2.  Attach black power leads to cassettes  
3.  Close lid to engage safety interlock.  Turn on power supply to 150V (40mA) constant 

Volts for 5-7 hours. 

 
 

 

 
 

Fig. 3-10 

 
 
 
 
 
 

 
 
 
 
 
 

 

Summary of Contents for DGGE-1001

Page 1: ...Denaturing Gradient Gel Electrophoresis Systems INSTRUCTION MANUAL DGGE 1001 DGGEK 1001 DGGE 2001 DGGEK 2001 DGGE 2401 DGGEK 2401 DGGE 4001 DGGEK 4001 DGGE 4801 DGGEK 4801 shown above DGGEK 2401 ...

Page 2: ...chniques 12 16 3 4 Vertical Gradient Formation 17 3 5 Preparation of the Cassettes 18 3 6 Buffer Cycling Connections 19 20 3 7 Running the Gels 21 3 8 Removing the Gels 22 3 9 Perpeniduclar Gel Casting 23 3 10 Perpendicular Gradient Formation 24 25 3 11 Buffer Siphon Pump Instructions for use 26 3 12 Maximum Well Comb Volumes 27 Section 4 Running Conditions 4 1 Recommended Power 27 4 2 Recommended...

Page 3: ...e highest practical standards of materials workmanship and design C B S Scientific warrants that the product has been tested and will meet or exceed published specifications This warranty is valid only if the product has been operated and maintained according to the instructions provided C B S Scientific warrants this product to be free from defects in materials and workmanship under normal servic...

Page 4: ... el usuario ni rendimiento insatisfactorio atribuible a circunstancias fuera del control de C B S Scientific C B S Scientific en ningún caso asumirá responsabilidad por daños incidentales o subsecuentes incluyendo en forma no limitativa la pérdida de utilidades de ingresos de oportunidades comerciales o del uso del producto y otros daños afines fuere cual fuere su origen ni por daños derivados del...

Page 5: ...ttes enabling the researcher to run up to 8 gels simultaneously Improvements to the technique include a simplified method for casting perpendicular and vertical gels using Gel Wrap single or dual gel cassettes have been redesigned eliminating the use of agarose plugs the need for an external peristaltic pump for buffer cycling has been eliminated bulky screw clamps have been replaced with polyprop...

Page 6: ...wer supply must enter the unit through the lid assembly providing a safety interlock to the user DC current to the unit is broken when the lid is opened Do not attempt to use the unit without the safety lid Always turn the power supply off before removing the lid or when working with the unit in any way Follow safety precautions specified by the power supply manufacturer Input Power Mains to safet...

Page 7: ...Gradient maker 20mls per side Cat GM 40 2 each combs 2 sets glass plates 2 pair spacers and 2 each Gel Wrap gaskets White spring clamps 12 each GPC 0002 177 and 16 each GPC 0001 177 Number of clamps received with system will vary depending on format of DGGE Buffer siphon pump with tubing Cat BSP 1000 DGGEK Series also includes EPS 300 or EPS 300 II Power supply Peristaltic Mini Pump for gel castin...

Page 8: ...eld is between the bottom side of the heater control unit and the TOP surface of the tank lid Small bore silicone tubing for buffer cycling is already attached to the manifold outlets Connect large bore tubing from bypass pump to barb fitting on plastic manifold on underside of lid WARNING Do NOT turn on Heater Stirrer until tank has been filled with buffer Heater Stirrer Bypass Pump Installation ...

Page 9: ...www cbsscientific com Tel 858 755 4959 8 3 1 Unit Set up and Unpacking Instructions continued 3 Install thermometer into red silicone stopper on lid Moisten before inserting See Figure below Fig 3 2 ...

Page 10: ... tank until the level of buffer reaches the underside of the upper reservoir The upper reservoir holds only 50mls 6 Attach the power supply to the safety interlock Refer to Figure below 7 Attach the safety interlock to a wall mains outlet 8 Plug the Heater Stirrer into a wall mains outlet 9 Do NOT attach power leads from power supply to safety interlock until gels are loaded and installed in the t...

Page 11: ...www cbsscientific com Tel 858 755 4959 10 3 1 Unit Set up and Unpacking Instructions continued 10 Turn Heater Stirrer On and set temperature to 60 C Figure 3 2 Fig 3 4 Temperature Setting 60 C ...

Page 12: ...ched glass plate to designate which side is the inside and outside The same side of the notched plate should always sit against the reservoir gasket A Gel Casting using Gel Wrap Gasket Casting method For Vertical DGGE use the set of spacers which do not have the small hole or channel milled into the lower end these are for casting perpendicular gels 1 Start by holding the rectangular back plate wi...

Page 13: ...lass 3 3 Gel Casting Techniques A Gel Wrap Gasket Casting method continued 4 Place the notched plate on top of the bottom assembly starting from the bottom edge and gently easing the plate down Verify the gasket is smooth around the edges and then clamp along the bottom 5 Lift the assembly and stand it on the base of the clamps For leveling push glass plate assembly down until it stops against cla...

Page 14: ... be fitted with a leur valve V 2 and a 20ga needle with attached tubing to deliver acrylamide to the gel plate sandwich as shown in figure 3 5 With the height differential between the leur valve V 2 and the top of the gel plate sandwich between 2 4 inches it takes approximately 5 9 minutes to cast the gel Raising the height of the gradient maker can increase flow rate Flow can be regulated by turn...

Page 15: ...aker GM 40 and a mini pump 1 Alternatively you may choose to use a mini pump or other peristaltic pump to cast gels as shown in fig 3 6 If so secure the gradient maker to a ring stand and connect the outlet tubing to the mini pump tubing adapter Connect tubing from mini pump to a 20ga needle for affixing between glass plates Fig 3 6 ...

Page 16: ...h alcohol 2 Place a separating sheet in the gel caster 3 Place the back plate non eared on the sheet 4 Place the 0 75mm spacers on each side of the back plate 5 Place the top plate eared on the spacers to form 1st sandwich Repeat as required Continue to Section 3 4 Vertical Gradient Formation Mini Pump Magnetic Stirrer Gradient Maker Multi Gel Caster Stopcock SC 2001 Silicone Tubing SCT 100 Fig 3 ...

Page 17: ... right side of gradient maker C 2 GM 40 fig 3 5 and open interior valve to allow air bubble to escape Let as much as 1 ml B solution BACKFLOW into left side of gradient maker Decant the 1 ml back into right side with pasteur pipette Remove any residual solution from C 1 with absorbent paper 4 Add solution A to left side of gradient maker 5 Turn on magnetic stirrer 6 Exit tube with needle should be...

Page 18: ...events perpendicular electrical fields from intenferring with outside lanes Re clamp to cassette using 1 clamps 4 per side Dislodge by pulling the bottom section of the Gel Wrap from the plates to allow buffer contact with the gel See figure 3 8 3 Immerse cassette into pre heated buffer tank fig 3 8 and attach recirculating tubing to each upper reservoir fig 3 9 a b c Rinse loading interface of ge...

Page 19: ...www cbsscientific com Tel 858 755 4959 18 3 6 Buffer Cycling Connections Buffer Cycling Connections Single Fig 3 9a Buffer Cycling Connections Two Single Cassettes ...

Page 20: ... 4959 19 Fig 3 9b 3 6 Buffer Cycling Connections continued Fig 3 9c 1 Attach small bore tubing to barb fitting on cassettes 2 Turn heater stirrer on to fill upper reservoir begin buffer cycling Buffer Cycling Connections Dual Cassette ...

Page 21: ...ples at 1 1 with neutral dye Load 5 10 ug Genomic DNA well or 1 2 ug cloned B globin well Determine concentration by O D 260 2 Attach black power leads to cassettes 3 Close lid to engage safety interlock Turn on power supply to 150V 40mA constant Volts for 5 7 hours Fig 3 10 ...

Page 22: ...move cassettes remove glass sandwiches from cassettes 2 Using a wedge plate separator cat WPS 100 pry plates apart and immerse gel plate in buffer tray Stain with EtBr 5ug ml for 5 10 minutes Lift floating gel on plate out of tray and rinse with ddH20 Flip over onto saran wrap and peal off glass plate View bands on transilluminator Electro Blot or photograph ...

Page 23: ...glass notch A small amount of agarose is needed in two places once the gel sandwich is placed on its side both places are located on the lower corner where the single well comb interfaces the spacer and on the other side of the spacer where it interfaces the gel wrap 4 Once the agarose has set insert the gradient maker outflow tube tipped with a 20ga through the Gel Wrap and into the channel figur...

Page 24: ...μl TEMED 2 Add to tube B 11 5ml of 60 7 5 80 μI 10 APS 5 μI TEMED 3 Pour solution B into right side of gradient maker GM 40 and open interior valve to allow air bubble to escape Let as much as 1 ml B solution BACKFLOW into left side of gradient maker Decant the 1ml back into right side with pasteur pipette Remove any residual solution with absorbent paper 4 Add solution A to left side of gradient ...

Page 25: ...ating tubes to each upper reservoir fig 3 8 3 9a b c Rinse loading interface of gel with tank buffer to remove non polymerized acrylamide or excess urea Rinse each well with buffer and fill upper reservoir 12 Load samples 13 Turn on power supply to 150V 40mA constant Volts for 5 7 hours 14 When run is completed turn off power supply disconnect recirculating tubes and power leads remove cassettes r...

Page 26: ...tions of your institution Fig 3 12 1 Attach pump assembly to corner of tank as shown above 2 Place the discharge tubing into a waste carboy located LOWER than the pump inlet 3 Begin siphon with half strokes until liquid starts to flow Leave shaft fully extended to allow siphon to operate 4 To stop siphon depress handle and remove pump from tank ...

Page 27: ...per tooth microliters ul 2 0mm thickness volume per tooth microliters ul 16 5 2 3 2 130 19 1 74 52 111 134 20 4 2 2 4 130 17 7 56 74 63 84 22 4 35 1 6 130 17 7 33 44 66 88 29 2 9 1 6 130 17 7 38 51 77 102 30 2 8 1 6 130 17 7 37 49 74 96 SECTION 4 Running Conditions 4 1 Recommended Power 1 150V 40mA CV for 5 7 hours 4 2 Recommended Buffers Warning Do not mix buffer in the DGGE tank STOCK SOLUTIONS ...

Page 28: ... PP 95 13 4 V C Sheffield D R Cox L S Lerman and R M Meyers 1989 PNAS 86 232 236 5 Guldberg P Henriksen K F and Guttler F 1993 Molecular analysis of phenylketonia in Denmark 99 of the mutations detected by denaturing gradient gel electrophoresis Genomics 17 141 146 6 Moyret C Thellet C Puig P L Moles J P Thomas G and Hamelin R 1994 Relative efficiency of denaturing gradient gel electrophoresis and...

Page 29: ...pose the apparatus or components to phenol acetone benzene halogenated hydrocarbon solvents or undiluted alcohol s Avoid prolonged exposure of the apparatus or components to UV light Do NOT treat with diethylpyrocarbonate DEPC treated water for extended periods at 37 C A brief rinse with DEPC water is sufficient after a thorough wash 5 2 Maintenance The following inspection and maintenance procedu...

Page 30: ...s and spacers DGGEK 1001 DENATURING GRADIENT GEL ELECTROPHORESIS SYSTEM 1 Dual Cassette 2 place Gel size 17 7cm w x 22cm l Includes Electrophoresis tank 1 dual cassette heater stirrer buffer cycler gradient maker 4 combs 2 each 1 well and 2 ea 16 well combs 4 sets of spacers 2 sets for vertical DGGE and 2 sets for perpendicular DGGE 2 glass plate sets 4 pieces gel wrap gasket 12ea 2 clamps and 18e...

Page 31: ...ell VGC 1022 1mm x 22 well VGC 1029 177 1mm x 29 well VGC 1029 1mm x 29 well VGC 1030 177 1mm x 30 well VGC 1030 1mm x 30 well VGC 1516 177 1 5mm x 16 well VGC 1516 1 5mm x 16 well VGC 1520 177 1 5mm x 20 well VGC 1520 1 5mm x 20 well VGC 1522 177 1 5mm x 22 well VGC 1522 1 5mm x 22 well VGC 1529 177 1 5mm x 29 well VGC 1529 1 5mm x 29 well VGC 1530 177 1 5mm x 30 well VGC 1530 1 5mm x 30 well VGC...

Page 32: ...0 243 4959 Fax 858 755 0733 Online ordering www cbsscientific com E mail address sales cbssci com Mailing address C B S Scientific Company P O Box 856 Del Mar CA 92014 Shipping address 10805 Vista Sorrento Parkway Ste 100 San Diego CA 92121 Credit Card Options Visa Mastercard Discover American Express ...

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