ELECTROPHORESIS OF DNA IN POLYACRYLAMIDE GELS

ELECTROPHORESIS OF DNA IN POLYACRYLAMIDE GELSGel SizesSmall: 165 x 130 mmMedium: 165 x 200 mmLarge: 165 x 260 mm5% Analytical Gels

Reagent	1 mm Small	1 mm Medium	1 mm Large
10X TBE Buffer (ml)	3	3.5	5
40% bis/acrylamide (ml)	3.75	4.4	6.2
Water (ml)	17.25	20.1	28.8
50% Glycerol (ml)	6	7	10
APS (mg)	34	40	57
TEMED (ul)	12.8	15	21.4
Total Volume (ml)	30	35	50

With other concentrations of a bis/acrylamide stock substitute:

25% bis/acrylamide (ml)	6.0	7.0	10.0
Water (ml)	15.0	18.5	25.0

30% bis/acrylamide (ml)	5.0	5.8	8.3
Water (ml)	16.0	18.7	26.7

50% bis/acrylamide (ml)	3.0	3.5	5.0
Water (ml)	18.0	21.0	30.0

5% BAC Cross-linked Polyacrylamide Gels

Reagent	1 mm Medium	1 mm Long	4 mm Small	4 mm Medium
10x TBE Buffer (ml)	8	10	16	20
Water (ml)	14	17.5	28	35
50% Glycerol (ml)	8	10	16	20
20% bac/acrylamide (ml)	10	12.5	20	25
APS (mg)	16	20	32	40
TEMED (ml)	200	250	400	500
Total Volume (ml)	40	50	80	100

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 Gels10 ml 7.5% Polyacrylamide Gel Solution65 µl 10% (w/v) APS 12.5 µl TEMEDSEQUENCING 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

RECIPES10X 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. (Biotechniques10:182, 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% Glycerol25 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