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Klenow fill-in of 5' overhangs to get blunt ends

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#1 neuropath



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Posted 26 May 2014 - 03:50 AM

I have been doing cloning for almost 20 years now and as far as I can tell, whenever there is a need to create a blunt end after digesting with a 5' cutter enzyme, protocols always recommend using Klenow to fill-in the 5' overhang. That was what I was taught when I first started working in the lab as a young, impressionable student. Recently however, I needed to blunt the ends produced by two different enzymes and then ligate them to recreate an open reading frame. I have Promega Klenow in my lab and I decided to read the product insert in detail. To my great surprise, I noticed this note for the first time after having read it almost 10 times over the past few years:


Note: Klenow Fragment, Exonuclease Minus, will leave a single-base 3´-overhang for a significant proportion of the DNA fragments during the fill-in reaction (8). Therefore, these fragments should not be used in blunt end cloning experiments.
Reference 8 is: Clark, J.M., Joyce, C.M. and Beardsley, G.P. (1987) Novel blunt-end addition reactions catalyzed by DNA polymerase I of Escherichia coli. J. Mol. Biol. 198, 123–7.
Imagine my shock. It's like discovering a birth mark on your backside after being alive for 40 years wacko.png .
So why wasn't this mentioned more obviously before? I only see the warning in Promega product info but not those from other major brands. Now thinking back, it explains why I sometimes get 1 extra bp at the ligated site. If recreating reading frame or restriction site isn't involved, then it is of no concern. 
Can someone please share their thoughts on this issue?

Edited by neuropath, 26 May 2014 - 04:49 AM.

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