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