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1. Add agarose to 1X TBE (or TAE) buffer. For gel size 20 x 24 cm, use 300-400
ml buffer and 0.7 to 1.0% agarose.
2. Melt agarose in 500 ml flask in microwave oven, mixing several times
during heating. Let cool to 55 C (until flask can be held).
3. Tape the ends of gel tray and place on a level bench.
4. Add ethidium bromide: 2.5 ul of 10 mg/ml stock per 100 ml. (gel cam
also be stained in ethidium bromide bath after electrophoresis (see point
9).
| NOTE: Ethidium bromide is mutagenic - wear gloves
when handling, and use extra caution. Change gloves when contaminated
and dispose in separate waste for ethidium bromide. |
5. Pour agarose into tray and insert combs. Remove bubbles with a pipette
tip. Allow to solidify.
6. Remove tape and place tray in gel boxes. Pour enough 1X TBE (or TAE)
buffer into the gel box to cover the gel by at least 0.5 cm. Remove combs
when ready to load samples.
7. Load 1 ug Lambda digested with Hind III (5 ul of 200 ng/ul stock) as
molecular weight marker, then load samples.
8. Run at 15-25 V for 12-24 hours.
9. If no ethidium bromide was added to the agarose: stain gel in 1 ug/ml
ethidium bromide (100 ul of 10 mg/ml ethidium bromide in 1000 ml dd H2O)
for 20 min. 10. Rinse gel for 20 min in 1000 ml ddH2O.
11. Slide gel onto UV transilluminator and take photo.
Photographing tip:
Place small piece of paper with writing or transparent ruler on the gel
to help focus.
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