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
| Reagent | |||
| 10X TBE Buffer (ml) | |||
| 40% bis/acrylamide (ml) | |||
| Water (ml) | |||
| 50% Glycerol (ml) | |||
| APS (mg) | |||
| TEMED (ul) | |||
| Total Volume (ml) |
| 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) | ||||
| Water (ml) | ||||
| 50% Glycerol (ml) | ||||
| 20% bac/acrylamide (ml) | ||||
| APS (mg) | ||||
| TEMED (ml) | ||||
| Total Volume (ml) |
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
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 Solution40% 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
