Southern blot nightmare

converrl at ucbeh.san.uc.edu converrl at ucbeh.san.uc.edu
Mon Nov 7 10:12:20 EST 1994


     I was writing regarding a problem I have with stripping a Southern Blot
from a Magnacharge NT membrane:

1. After probing this genomic Southern with a 2kb nick translated probe (100
microliters of 3*10^5 cpm/microliter probe), my exposure shows that probe is
hybidizing all over the blot in a random pattern (it looks like an inkblot
test!). My boss told me that this type of background comes from not filtering
the probe through a .45 micron Polysulfone acrodisc syringe filter prior to
hybridization.

2. I have never encountered background like this before and do not make a habit
of filtering my pobes.

3. After Stripping this filter and reprobing it, I got the exact same "inkblot"
pattern as before. My boss seems to feel that this is protein &/or other
contamination that will not come off no matter how extensively I strip. My
stipping conditions are: 70 degrees Centigrade, .05 N NaOH, 0.1X SSC, 1% SDS. I
change this 2 times and wash 30 min. each time (we have one of those rotating
hybridization ovens).

     My questions are the following:

1. Is the assesment that I did not filter my probe the correct one for the
source of all this background?

2. Is there any way I can strip the "junk" off this blot without removing the
DNA?

                      E-mail any helpful suggestions!!

                      Richard



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