Posi blot transfer

Preparation of gel for Posi blot transfer

1、Cut gel to size of preexisting mask or cut new mask.

2、For depurination: put gel in pyrex dish, add 500ml .25N HCl (10mL conc. HCl+450ml H₂O) and shake 15min.

3、Remove HCl, rinse w/H₂O

4、Add 500ml 1X Posiblot denaturing solution and shake 15min.

5、Repaeat #4

6、Remove denaturing solution

7、Add 500ml Posi blot neutralizing solution and shake 15min.

8、Repeat #7

9、While gel is in 2nd neutralizing wash (step 8):

a) make up 1 liter 5XSSPE

b) cut gene screen plus membrane to size of gel and mark membrane w/pen

c) cut whatman larger than size of gel

d) soak gene screen plus and sponge in 5XSSPE

Comments: see reagents for recipes for Posiblot neutralizing and denaturing solutions.