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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
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 |
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
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| 50% bis/acrylamide (ml) |
16
|
28.8
|
40
|
| 10x TBE Buffer (ml) |
10
|
18
|
25
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| 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:
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
Send comments and updates to
Dr. Bart Frank, Arthritis and
Immunology Program, OMRF
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