Tandem repeat from SiteDirectedMutagenesis

Paul Shinn pshinn at force.stwing.upenn.edu
Thu Jul 29 13:42:31 EST 2004


   After sequencing several clones using the QuickChange protocol
I am seeing a tandem repeat of my mutagenic primers.  I have used
this before and this is the first I've seen this happen so I worry
it's a primer batch problem but I can't explain why.

   I'm following the Stratagene QuickChange protocol but not using the
kit.  I can't use their kit because their comp cells aren't compatible
with my insert.  I am using KOD HiFi proof reading enzyme which is much
more processive than the Pfu in the kit and the primers are made in house
but not HPLC or gel purified.  However, they are run through a Sephadex
G-50 column to get rid of some of the smaller n-1 primers.

   In independent experiments to verify how full length my oligos are
after synthesis, I have found that >80% of the cloned inserts using 
43mers are full length so I don't feel that anything is wrong with the 
oligos.

   I have seen one other post where the user had this happen but I didn't 
see how he got around it.

				Thanks, Paul



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