blunt cloning of PCR fragments

Gregor Bucher st2153 at zi.biologie.uni-muenchen.de
Wed Aug 2 11:55:50 EST 2000


HI MIKE!

In our lab they say that it depends on the primer sequence if you will get
A-overhangs or not. And as nobody knows what conditions lead to one or the
other end, we always use Klenow and subsequent PNK to get blunt and
phosphorylated ends.

Gregor

>I've been trying to blunt-end clone some PCR fragments without much
>success.  Controls lead me to beleive that there isn't  problem with the
>ligase or ligation condition.  The PCR fragments were generated using
>KlenTaq LA from Sigma.  Since the enzyme mix is supposed to have a 3' to 5'
>proofreading activity, I figured the fragments should be coming out of the
>reaction with blunt ends.  Am I wrong about that?  Should I go ahead and
>polish up the ends in a separate step anyway?  Thanks for your input.
>
>Mike
>
>Michael L. Sullivan, Ph.D
>
>U.S. Dairy Forage Research Center
>1925 Linden Drive West
>Madison WI, 53706
>
>(608) 264-5144 Phone
>(608) 264)-5147 Fax
>
>
>---
-- 
Gregor Bucher, Zoologisches Institut der LMU München
0049/89/5902/444
st2153 at zi.biologie.uni-muenchen.de
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