Tandem repeat from SiteDirectedMutagenesis

Sergio yokese at hotmail.com
Thu Jul 29 20:28:04 EST 2004


Paul Shinn 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

Does KOD HiFi have 5'->3' exonuclease activity?
since you are amplifying a circular molecule, nick translation during 
the PCR may produce different products. Those products may generate a 
product with duplications of the primer annealing region....
I've tried to draw an scheme in ASCII but is kind of difficult. I'd 
suggest to draw it by yourself to see how it can happen.
Besides, we noticed that adding ligase to the final product, in a naive 
attemp to seal the nicks and improve efficiency, actually generates a 
lot more of undesired products that otherwise wouldn't be functional 
once within the cell.

Sergio



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