Posi blot transfer
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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.