5' filling with Klenow

Martin Kennedy mkennedy at chmeds.ac.nz
Mon Sep 12 19:59:48 EST 1994


In article <1994Sep13.124605.594 at chmeds.ac.nz>, mkennedy at chmeds.ac.nz (Martin Kennedy) writes:
> In article <35289a$te at mark.ucdavis.edu>, ez003450 at chip.ucdavis.edu () writes:

Ooops.......
> 
> I would definitely sequence the junction.  Don't forget that Klenow still 
> has a 3'->5' nuclease activity, and it can remove part of the 5' overhang.  
                                         ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^
On second thoughts this isn't a sensible explanation for the occasional 
losses of bases seen at these sites.  Perhaps it is caused by 5'->3' 
nuclease present as a contaminant in some component (DNA prep, enzyme etc)
of  either the digestion or fill-in reaction.  Whatever the cause, loss 
of bases can occasionaly occur after these sorts of manipulations, and it 
would be wise to sequence.
  

-- 
Cheers,

Martin

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