Protocol
Polyacrylamide Gel Electrophoresis Sequencing Gel.
(for DNA polymerase kinetics)
Conditions
20% acrylamide (19:1 acrylamide:bis-acrylamide)
1 x TBE buffer 7 M urea
2000 V
preparation of gel
| |
gel thickness |
|
Compound |
0.4 mm |
0.4 mm |
0.8 mm |
1.6 mm |
|
| urea |
28 g |
33 g |
50 g |
80 |
|
| 40% acrylamide solution |
25 mL |
30 mL |
45 mL |
72 mL |
|
| 10 x TBE |
6.7 mL |
8 mL |
12 mL |
20 |
|
| water |
16 mL |
19 mL |
29 mL |
45 |
|
| TEMED |
40 mL |
45 mL |
60 mL |
100 mL |
|
| APS (10%) |
300 mL |
400 mL |
600 mL |
1000 mL |
|
| Total volume |
67 mL |
80 mL |
125 mL |
200 mL |
|
- Mix urea, acrylamide solution, TBE and H2O. Stir solution.
Mixture cools down as urea dissolves. May quicken dissolution by
warming solution in crystallization dish with warm water from
tap.
- Clean and dry glass plates. Coat sides with 0.5 mL Sigmacote
every 10 gels
- when urea has dissolved add TEMED and APS
- Pour gel. For kinetics we typically use the 0.4 mm
thick spacers. We use the thicker spacers for the
purification of oligodeoxynucleotides.
- Let sit until polymerization is complete.
Solutions
Acrylamide solution
40% acrylamide, 19:1 acrylamide:bis-acrylamide Prepare from scratch
| acrylamide |
380 g |
| N,N-methylene-bis acrylamide |
20 g |
| add water up to 1 L. |
|
Acrylamide is neuro-toxic. Weigh acrylamide in
hood. Wash surfaces with water.
store in refrigerator.
Purchase from Fisher:
1 L catalog number. BP14061 list price $180.
10% Ammoniun persulfate (APS)
| ammonium persulfate |
1 g |
| add water up to 1o mL. |
|
| store in refrigerator |
|
10X TBE
0.89M Tris, 0.89M boric acid, 0.02 M Na2EDTA
| Tris base |
121 g/mol |
108 g |
| boric acid |
61.8 g/mol |
55 g |
| H4EDTA |
292 g/mol |
5.8g |
| NaOH |
40 g/mol |
1.6 g |
| Fill to 1 L with water pH should
be 8.3 |
|
|
|
