Our gel rigs and supplies are from CBS Scientific.
The National Diagnostics Website has very helpful background on RNA/DNA polyacrylamide gels.
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.
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.
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.