Can I successfully blunt it?

[Helge Weissig]

In article <2sgptr$7ej at dingo.cc.uq.oz.au>, andrew at qimr.edu.au (Andrew
Healey) wrote:

> Huanquan Zheng (zheng at cc.Helsinki.FI) wrote:
> : I have an insert which prosess both 5' and 3' overhang protruding in
> : upstream, to blunt this insert, the following has been planed to do:
> 
> : 20 ul reagent: 
> : Klenow fragment enzyme 3U
> : Insert		       3g
> : dNTPs		      2mM final conc (high?)
> : 10x klenow buffer     2ul
> 
> : 30 min at RT
> 
> : Can I successfully fill in recessed 3' terminus in downstream and
> : remove that 3' protruding in upstream after above treatment?
> 
> Yes, sure. T4 Polymerase is also OK and somewhat more efficient. The dNTP's
> *are* way too high...that's the concentration of the stock mix.....use one
> in 10 of this (giving 200 uM final).
> 

hmmm... would it not be better to use T4 Polymerase instead, since Klenow
really only fills in the 5' ends, whereas T4 also chews off the 3' ends?

and: if Klenow, then don't use too much. One unit should do, otherwise you
end up with 3' termini due to its 3'-5' exonuclease activity.

my 0.02 units worth
cheers,
Helge


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Helge WEISSIG                              e-mail: helgew at ljcrf.edu
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