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
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1 mm Large
Reagent
1 mm Small
1 mm Medium
10X TBE Buffer (ml)
3
3.5
5
40% bis/acrylamide (ml)
3.75
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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 el ectrophorese 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)
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40
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
10x TBE Buffer (ml)
10
18
25
10% (w/v) APS (ml)
0.668
1.2
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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 for m.)
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 foi l 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 mi cron filter and store in foil wrapped bottle.

7.5% Polyacrylamide Gel Solution
100 ml
40% Bis/Acrylamide Stock Solution 18.75 ml 37.5 ml
10x TBE buffer 1 0 ml 20 ml
50% glycerol 20 ml 40 ml
Water 51.25 ml 102.5 ml
TOTAL VOLUME 200 ml

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Copyright 1993, 1996, 1997 and 1998 by Mark Barton Frank, Ph.D.
Proper citation for data acquired from this document is: "Frank, M. B. Electrophoresis of DNA in Polyacrylamide Gels. In: Frank, M. B. ed. Molecular Biology Protocols. (http://omrf.ouhsc.edu/~frank/acryl.html). 1997. Oklahoma City. Revision Date: September 17, 1998."

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