APPENDIX 6 - POLYACRYLAMIDE GEL PREPARATION

Apparatus Setup - Part I

1. Scrub four 8cm x 18cm glass plates thoroughly with non-ionic detergent (such as Decon 90) with a scrubbing brush and rinse

2. Clean one side of each plate, both sides of spacers and well combs with 70% ethanol

3. Place spacers between plates and place plates in clamps, making sure that plate edges and spacers are all level with the clamps

4. Secure plates and clamps onto gel caster with black cams

Gel Preparation (5% Gel)

5. To a 50 mL falcon tube add:

6. When ready, add: 7. Pour gel between the plates

8. Place well combs in top of gel

9. Monitor for ~20 minutes to make sure gel does not leak

10. When gel has set, remove combs carefully and immediately wash wells with 1x TBE to prevent small amounts of acrylamide polymerising and causing uneven DNA migration

Apparatus Setup - Part II

11. Remove cams and place the upper buffer chamber over the plates. Secure with cams.

12. Fill upper and lower buffer chambers with 1x TBE so bottom of gel and upper electrode are covered with buffer

13. Put lid on and connect electrodes

Running the gel

14. Load DNA with ~2 mL of gel loading dye

15. Run the gel at 150V - 200V for ~3/4 hour