Tandem repeat from SiteDirectedMutagenesis

Tom Vink tvink at xs4all.nl
Fri Jul 30 02:18:27 EST 2004


Hi,

We have seen this also once with one oligo and got around it by
raising the annealing temp to 68 C. Hope this helps.

Grtz Tom

On Thu, 29 Jul 2004 18:42:31 +0000 (UTC), Paul Shinn
<pshinn at force.stwing.upenn.edu> wrote:

>   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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