oligo Southerns...HELP!

Hanson Lab Lab_Mac_Hanson at QMrelay.mail.cornell.edu
Fri Jun 17 08:27:05 EST 1994


In article <1994Jun16.134322.1 at opal.tufts.edu>, kmorris1 at opal.tufts.edu
wrote:

> I hope someone will be able to help me... I'm going crazy with this problem!
> I am trying to set up a competitive PCR to detect mutant and wild-type alleles
> of p53 in a particular cell line.  I have made both WT and M competitor
> constructs, and the PCR itself seems to be working well.  Both alleles from
> genomic DNA, however, amplify the same size fragment so I have to use
> differential hybridization with specific oligonucleotides to quantitate. 
[stuff deleted]
I had a similar problem with oligo Northerns using 2 19mer oligos that were
identical except for two bases (As in one, Gs in the other).  Since my
targets were RNAs containing Us or Cs opposite these base differences, I
had a terrific problem with the G oligo hybridizing to both (G-U vs G-C). 
Same problem as you, no difference in the melting behavior of the hybrids,
couldn't differentially wash off the G-U pairs by incrementally increasing
wash or hyb temp.  I successfully got around this problem by doing the
hybridization of the G-containing oligo in the presence of 10X cold
A-containing oligo.  It worked for me--I would suggest trying competitive
hybridization like that.
Good luck--
Claudia Sutton
Genetics and Development
Cornell University
cas9 at cornell.edu



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