Polyacrylamide Gel Electrophoresis (PAGE)
for use withRibonuclease Protection Assay (RPA):
1. Making the Gel:
5% Denaturing gel for Ribonuclease Protection Assay:
Urea,high quality 7.2 g
10X TBE 1.5 ml
30% acrylamide/bis 2.5 ml
or use ready to go 40% acrylamide 1.9 ml
Deionized H2O up to 15 ml
Stir at room temperature until urea has dissolved then add:
10% APS 120 ul
TEMED 16 ul
For an 8% gel use 4 ml of 30 % acrylamide (3 ml of 40%)
For a 10% gel use 5 ml of 30% acrylamide (3.75 ml of 40%)
2. For denaturing gels only, heat all tubes for 3-4 minutes at 90’C.
3. Vortex, spin down and put on ice.
4. Pre run gel for 5 minutes, rinse out well with buffer then immediately load gel.
5.Run for approximately 1 hour at 250 volts in TBE buffer.
6. Transfer to positively charged nylon membrane according to manufactures protocol.
Recommended % acrylamide: Size of Bands: Bromophenol Blue:
(Denaturing gels)
4 >250 30
6 60-250 25
7 40-120 20
10 20-60 10