Southern blot nightmare

vitor warwar vwarwar at unlinfo.unl.edu
Wed Nov 9 00:22:56 EST 1994


converrl at ucbeh.san.uc.edu wrote:
:      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!!

     Dear Richard:

     1. I do not think that cleaning the probe will do any good. I
never clean probes, unless I have to do a "very" low stringency
hybridization.

     2. Your problem may be due to the salmom sperm DNA that you
may be using in your hybridization solution. Check its concentra-
tion and if its is not more than 200-300 base pair in lengh.

     Good luck,

     Vitor Warwar
     <vwarwar at unlinfo.unl.edu>

--
______________________________________________________________________
     Vitor Warwar / 406 Plant Sciences Hall / University of Nebraska
     Lincoln NE 68593-0722 / USA / vwarwar at unlinfo.unl.edu
_______________________________________________________________________



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