How many bases overlap in recombinant PCR?

brett brett at BORCIM.WUSTL.EDU
Tue Oct 8 11:25:42 EST 1996


>Hi,
>
>   I'm doing recombinant PCR to insert a fragment of 30 bps into a known
>1kb sequence.  Of course, in an effort to minimize costs, I assumed that 5
>bp's overlap would be quite enough.  What is the minimum number of bp's
>that still works nicely?  Has anyone tried with just 5 bp's over a total
>final PCR product of nearly 1 kb?  Thanx for any info....
>
>Barry Mook
>barry at sccs.swarthmore.edu

I have used 9bp in each direction with success. 5bp maybe getting kinda short.
Assuming 50%GC, a 5bp overlap would have Tm ~30C. Its worth a try, but you may
have to play with your annealling conditions a bit (Temp, Mg++, etc.) Good luck.


Brett Lindenbach
    
Program in Immunology                              
Washington University - St Louis                  
brett at borcim.wustl.edu                             

"I own my own pet virus. I get to pet and name her." - Cobain




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