Does Klenow proofreading multiple-base 3' overhangs or just single base?

brett at BORCIM.WUSTL.EDU brett at BORCIM.WUSTL.EDU
Fri Mar 10 18:03:33 EST 1995


>Can anyone tell me if the Klenow proofreading activity used for trimming off 
>the extra 3' nucleotide added during PCR RXNs also works for trimming back
>multi-base 3' overhangs of restriction-digested fragment ends?  I've come 
>across a number of papers which state in their M&M that a digested fragment 
>was "blunted" with Klenow.  Since you can't "fill-in" in front of the 5' 
>recessed end, they must mean the use of Klenow to nibble back.
>Regards, Peter 
>**********************************************************************
>*  Peter M. Muriana, Ph.D.             317-494-8284   TEL            *


Klenow's 3'->5' exo activity is rather low. It would be better to use
T4 DNA Pol for this. So, to your restriction rxn (10ul), add 0.5ul of
a cocktail containing 2.5mM ea dNTP, and a coupla units of T4 DNAP per
ug DNA. Cook for 15-20' @ 11C, heat kill, if desired. This general mix
will work for both chewing back as well 3' and filling in 5' with great
efficiency. Another option is to chew back with Mung Bean, etc. but that's
more work, and unless you need to chew back a 5' overhang, not worth the
extra effort (often having to change buffers). 



Brett Lindenbach
Lucille P. Markey Student in Human Pathobiology
Program in Immunology
Washington University - St Louis
brett at borcim.wustl.edu





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