klenow and ligations

Ned Mantei mantei at cell.biol.ethz.ch
Mon Apr 16 06:55:34 EST 2001


In article <B70093D9.4ED%roee.atlas at weizmann.ac.il>, 
roee.atlas at weizmann.ac.il (Roee Atlas) wrote:

>Hi,
> 
>I'm doing ligation of blunt DNA. To blunt the DNA I'm  using a Klenow
>fragment. Is it possible to klenow the DNA fragment after gel excision, 
>then
>heat-inactivate the Klenow enzyme and put the blunted fragment straight to
>ligation? Do the dNTP's in the reaction disrupt the ligase?
>

My experience has been that it's much better to do the fill-in just 
after the restriction digestion. Klenow works in the usual restriction 
enzyme buffers, so you can just add dNTPs (to ca. 20 uM) and Klenow (to 
about 40--80 units/ml) and incubate 15 minutes at room temp (not 37 C).I 
then do a phenol extraction and ethanol precipitation before the agarose 
gel. We had the strong impression that heat inactivation was not good 
enough, and that the enzyme was either not totally inactivated or was 
renaturing during the ligation reaction.

-- 
Ned Mantei
Department of Cell Biology, Swiss Federal Institute of Technology
CH-8093 Zurich, Switzerland




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