Help!!!! how to clone a PCR fragment?

Mike Dalrymple dalrymple at pplros.demon.co.uk
Fri Aug 4 05:31:00 EST 1995


In article
<Pine.AUX.3.91.950803180003.17417A-100000 at mail.med.cornell.edu> Heidi
Moss, hmmoss at MAIL.MED.CORNELL.EDU writes:
>Subject: Help!!!! how to clone a PCR fragment?
>From: Heidi Moss, hmmoss at MAIL.MED.CORNELL.EDU
>Date: 3 Aug 1995 15:29:13 -0700
>

I have never actually performed this experiment as you describe, however,
I have experience of some of the steps you outline.

A.	I do a one tube reaction which "polishes" and then kinases. The PCR
fragment
is isolated from a gel and incubated first with Klenow and dNTPs (I
believe
Klenow has a weak 3'-5'exonuclease activity, anyway it works!), followed
by
addition of ATP and T4 PNK. This method was published in Biotechniques
more than
2 years ago, sorry I've lost the reference.

B.	The only other pearl of widom I can add is that you may have problems
cloning a Sticky-blunt fragment. We did. We decided to follow the ligation
by gel and saw all the stickies join very quickly leaving blunt-blunt
intermediates.
There was none of the "correct" intermediate. We then decided to lightly
phosphatase
the "insert" fragment to prevent the stickies joining and that seemed to
work
but not very efficiently.

Hope this is of some help. If you want the precise buffer components for
the
polishing, email me.

Mike Dalrymple



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