SSCP single strands

Your Name Here labuser at waikato.ac.nz
Sun May 18 21:05:46 EST 1997


  I am using SSCP to analysis for mutations in exons 5-8 of the p53 gene. 
I am denaturing my PCR product (1:2) with 95% formamide, 10mM NaOH, 0.5%
of the dyes BB and XC, for 5minutes at 95 degrees C, followed by plunging
the tubes in a 4 degree C water bath.  

  I am seeing plenty of double stranded PCR product on a 6% polyacrylamide
gel, however the amount of single stranded product is variable and
sometimes there is none.
   
I would be grateful for any emailed advice regarding how to achieve
efficient denaturation (without methyl-mercury or biotin) of my PCR
product for SSCP analysis.

   Thanks in advance,

   Greg Jacobson

    gmj at waikato.ac.nz

   Greg



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