southern blot lower limit

Bill Alexander alexanderw at cber.cber.fda.gov
Tue Apr 23 14:26:36 EST 1996


In Article <4liq3j$idg at service2.uky.edu>, "Michael W. Thompson"
<mthom0 at pop.uky.edu> wrote:
>            I have been having the same problem with a southern blot similar to

>the one that you are working on.  The problem is, that I know this 
>sequence exists in the human genome.  I think the problem here is that 
>my probe is too small for the conditions I have used so far.  

15 to 18 bp is usually enough depending upon the complexity of the DNA being
probed.  Do the math: 4 to the Nth power vs complexity (usually the size) of
the genome being probed.  Repetitive sequences can mess this up.

>Have you 
>checked your probe by running it on a gel?  This might give you some 
>insight as to why you're not seeing your band.  Just a couple of 
>negative experiments isn't really enough to conclude that your target 
>sequence does not exist in the genome of the organism you're looking at.  
>What is your sensitivity?  10 ug of a positive control is an awful lot.  
>Maybe you should run a much smaller amount of your positive control to 
>make sure?  You should be able to detect down to about 1 pg of your 
>control in order to pick up a homologous sequence on you Southern blot.
>
>Hope all this rambling helps...
>
>Michael Thompson
>Dept. of Biochemistry
>University of Kentucky
>
How about trying to amplify out your target using PCR.  I came into the
middle of this thread but if you have one primer you can match it with an
Alu primer (or another repetitive seq.) and try to amplify out your target.  
Regards,

Bill Alexander
alexanderw at cber.cber.fda.gov

     "640K ought to be enough for anybody." -- Bill Gates, 1981



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