Sequencing PCR Products - Please Help

rs_santhosh at hotmail.com rs_santhosh at hotmail.com
Sun Jul 7 06:05:21 EST 2002


> I have been purifying PCR products with a Qiagen kit in
> anticipation of sequencing them.  However, when I run the
> DNA on an agarose gel to estimate the concentration, I still
> see primer-dimers.  I understand that primer-dimer
> contamination will ruin the sequencing reaction results.
> 
> Surely I am not the first person to have this problem.  I am
> working on using PCR conditions which use less primer and
> more template DNA.  I would rather not gel-purify each PCR
> product because I need to sequence so many of them.  Also, I
> think I will try a hot start for my PCR because I've read
> that it decreases primer-dimer concentration.
> 
> Does anyone have suggestions?  Has anyone tried sequencing a
> PCR product with some small primer-dimer contamination
> visible on the gel?  Any help would be much appreciated.
you see the quiagen catalogue there you will see the concentration of NaCl which elutye the fragments.first you wash the column with low concentration of NaCl, then further you wash with the eluate buffer provided by quiagen along with kit
http://www.biowww.net/index.php/forum/messagelist/7/

















More information about the Methods mailing list