ligation of PCR products

brett brett at BORCIM.WUSTL.EDU
Mon Oct 7 10:20:27 EST 1996


>Hi there,
>
>just to make sure: am I right in assuming that PCR products have no 5' 
>phosphate groups, and hence need kinase treatment before they can be ligated 
>to each other?
>
>can both be done in the same reaction (tube)?
>
>thanks a lot!
>
>alois.

Unless you phosphorylated your oligo, the PCR products will not ligate. One
problem with your strategy is that many PCR buffers contain NH3 ions, which
essentially kills kinase reactions. If this is not the case for you, it
will probably work, otherwise clean up your products and resuspend them in
kinase buffer. Furthermore, depending on which polymerase you used for
primer extension, there may be single base overhangs (usually dA) which
will
obviously interfere with your ligation. 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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