13 Sequencing
201
• plastic wrap
• syringes
• clamps
• tape
• 0.45 J..lffi disp. f:tlter
Procedure
1. Prepare a stock solution of 40o/o acrylamide/bisacrylamide:
Acrylamide
Bisacrylamide
Ultrapure water
190 g
10 g
ad 11
Add 20 g Amberlite MB1 and stir for 30 min to deionize, f:tlter through a
0.45 J..lm disp. fllter, keep at 4°C in the dark (aluminium wrapped) for up
to 1 week. Always use fresh solutions for gels.
2. Clean the glass plates using detergent, and rinse thoroughly with water.
Dry plates, and rinse again with ethanol.
3. Treat one side of each plate with Methyl-Silane™ (wearing gloves and
working in a fume hood). Rinse the plates thereafter with distilled water.
This step is essential to prevent the gel from sticking to the plates later.
4. Place 2 spacers on each side of the large plate and place the small plate on
top, both treated sides facing inward, thus forming a casting frame.
5. Tape the plates on both sides and the bottarn thoroughly, taking special
care on the corners, and clamp on both sides using several clamps.
6. Set the plates at a 30° angle with the top of the plates elevated.
7. In a beaker, mix:
40% acrylamide/bisacrylamide
Urea
10 x TBE buffer
Ultrapure water
15 ml
42 g
10ml
ad 100 ml
ln advance
Gelcasting