Polyacrylamide Gel Electrophoresis
Our gel rigs and supplies are from
CBS Scientific.
The
National Diagnostics Website has very helpful background on RNA/DNA polyacrylamide gels.
Pouring the Gel
For denaturing urea gels, we use the
SequaGel system. Check out the link to determine how to mix up a gel of the proper percentage.
| Spacer Width |
Gel Height |
Gel Width |
Gel Vol |
TEMED Vol |
10% APS Vol |
| 1 mm |
28 cm |
16.5 cm |
50 ml |
20 µl |
400 µl |
| 1 mm |
14.5 cm |
16.5 cm |
25 ml |
10 µl |
200 µl |
TEMED is N,N,N',N'-tetramethylethylenediamine. APS is ammonium persulfate. Together, they create the radicals to initiate polymerization of the gel. TEMED and 10% APS should be stored at 4°C. APS solutions should be freshly prepared for best results.
Acrylamide is a neurotoxin before it is polymerized. You are working with it in a liquid solution where spills may happen. Wear gloves, a lab coat, and safety glasses when working with polyacrylamide gels.
Loading the Gel
Formamide sample loading buffer.
Be sure to wash urea out of the wells using a syringe before loading the gel.
| Spacer Width |
Gel Width |
Well Height |
Well Width |
# Wells in Comb |
Max Sample Vol |
| 1 mm |
16.5 cm |
15 mm |
8 mm |
12 |
120 µl |
| 1 mm |
16.5 cm |
15 mm |
4 mm |
20 |
60 µl |
| 1 mm |
16.5 cm |
15 mm |
2 mm |
30 |
30 µl |
For sharp bands, you should load to much less than the maximum well volume.
Running the Gel
Generally denaturing polyacrylamide gels are run at a constant electrical power (Watts). This maintains a certain heated gel temperature during the run. For the 16.5 cm width gels, use 25 W.