GEL TRANSFER

1. Transfer gel to a tray containing ~250 mL of denaturing solution.  Gel needs to be covered by the

Solution.  Shake gently for 25 minutes.

2. Decant denaturing solution and replace with fresh denaturing solution.  Shake again for 25 minutes.

3. Decant denaturing solution;  rinse gel with ddH2O, slosh gently, rinse again with ddH2O, decant, and

Add ~250 mL of neutralizing solution.  Shake for 15 minutes.

4. Decant neutralizing solution; replace with fresh neutralizing solution; shake again for 15 minutes.

5. Decant neutralizing solution; rinse gel with ddH2O, slosh gently, rinse again with ddH2O, decant, and

Add ~250 mL of 20X SSPE;  shake again for 30 min.

6. Soak the following while gel is shaking in SSPE:

           1 transfer membrane in ddH2O

           2 thin blotting papers in 10X SSPE

           1 thick blotting paper in 5X SSPE

7. Remove and invert the gel on saran wrap so that the bottom of the gel is facing up.

8. Remove the bubbles between the gel and saran wrap.

9. Cover the gel with the transfer membrane that was soaked in ddH2O.  Remove ALL of the bubbles

Between the gel and membrane.

10. Cover with the 2 thin blotting papers soaked in 10X SSPE.

11. Cover with thick blotting paper soaked in 5X SSPE.

12. Cover with a stack of dry blotting paper, tray, and bottles to provide weight.  Let stand overnight.