Blotting of PFGE

Dr. Richard V. Goering rgoeri at creighton.edu
Tue Jul 23 10:55:20 EST 1996


As noted in previous posts, depurination is EXTREMELY important as you
would expect, given the size of the fragments you are trying to transfer.
The following protocol has routinely worked well for us:

depurination:

0.25M HCl - 1 hour with exchanges every 15 - 20 min. 

denaturation:

1.5 M NaCl + 0.5 M NaOH - 1 hour with 1 exchange at 30 min.

neutralization:

1.5 M NaCl + 1 M Tris-HCl (pH 8) - 1 hour with 1 exchange at 30 min. 

We then vacuum blot in 2 X SSC with excellent (ca. 100%) transfer in 30
min.

Dr. Richard Goering
Dept. Med. Microbiol. and Immunol.
Creighton Univ. Sch. Med.
Omaha, NE
USA




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