ELECTROPHORESIS OF DNA IN POLYACRYLAMIDE GELS

ELECTROPHORESIS OF DNA IN POLYACRYLAMIDE GELS

Gel Sizes
Small:             165 x 130 mm
Medium:         165 x 200 mm
Large:            165 x 260 mm

5% Analytical Gels

5% BAC Cross-linked Polyacrylamide Gels 

Pre-run gels for 30 minutes at 80V. Rinse the wells with running buffer. Load and electrophorese samples at 80V until dyes separate, then boost to up to 150 V. Medium sized polyacrylamide gels can be run overnight at 55-60 V to see all phi-X/Hae III cut molecular weight standards on the gel.


7.5% Mini (10 x 8.2 cm) Polyacrylamide Gels

• 10 ml 7.5% Polyacrylamide Gel Solution
  

• 65 µl 10% (w/v) APS 
  

• 12.5 µl TEMED
  
  
  SEQUENCING GELS (8% Polyacrylamide, 8 M Urea)
  <

  Reagent	40 cm gel	80 cm gel	1 m gel
  Urea (g)	48	86.5	120
  Water (ml)	37	66.5	92.4
  50% bis/acrylamide (ml)	16	28.8	40
  10x TBE Buffer (ml)	10	18	25
  10% (w/v) APS (ml)	0.668	1.2	1.67
  TEMED (ml)	50	85	118
  Total Volume (ml)	100	180	250

  Pre-run sequencing gels at 1800 V for 30 minutes. 
  
  With other concentrations of a bis/acrylamide stock substitute:
  

• 30% bis/acrylamide(ml)		48	66.7
  Water (ml)		47.3	65.7

  
  

• 40% bis/acrylamide (ml)		36	50
  Water (ml)		59.3	82.4

  ---

  RECIPES
  
  

  • 10X TBE (1M Tris, 1M Boric Acid, 20mM EDTA, pH 8.3)

  • 242.2 g Tris

  • 123.66 g boric acid

  • 14.89 g EDTA

  • Adjust pH to 8.3. QS to 2 liters. Autoclave. (Biotechniques 10:182, 1991 claims that filtering up to a 20x TBE solution through 0.2 - 0.45µ cellulose acetate or cellulose nitrate filters prevents formation of precipitants during long-term storage. The solution may be reautocalved to dissolve precipitates that form.)
    

  • 50% Glycerol

  • 25 ml 100% glycerol

  • 25 ml water

  • Autoclave. Concentrated glycerol is quite viscous. Be sure all of it has been transferred from the stock bottle. 
    

  • 30% Acrylamide Solution (0.8% bis)

  • 60 g acrylamide

  • 1.6 g bis

  • Heat to dissolve in approximately 100 ml of water. QS to 200 ml with water and filter with Whatman #1 filter paper into a foil wrapped bottle. 
    

  • 40% Acrylamide Solution (19:1 acrylamide:bis)

  • 80 g acrylamide

  • 4.21 g bis

  • Heat to dissolve in approximately 100 ml of water. QS to 200 ml with water and filter with Whatman #1 filter paper into a foil wrapped bottle. 
    

  • 50% Acrylamide Solution (19:1 acrylamide:bis)

  • 237.5 g (97%) acrylamide

  • 12.5 g bis

  • Dissolve in 250 ml hot water. Filter to remove debris and wrap in foil.
    

  • 20% Bac-Acrylamide Solution     WARNING: wear gloves

  • 100 ml solution

  • 18.98 g acrylamide

  • 1.02 g BAC (N',N'-bis-acrylylcystamine)

  • Heat approximately 75 ml of water to almost boiling. Add acrylamide and cover with a watch glass. Mix briefly to dissolve and reheat to near boiling. Add BAC. QS to 100 ml with water. Do not autoclave. Filter sterlize with Nalgene 0.45 or 0.2 micron filter and store in foil wrapped bottle.
    

  
  7.5% Polyacrylamide Gel Solution
  

• 40% Bis/Acrylamide Stock Solution	18.75 ml	37.5 ml
  10x TBE buffer	10 ml	20 ml
  50% glycerol	20 ml	40 ml
  Water	51.25 ml	102.5 ml
  TOTAL VOLUME	100 ml	200 ml