Cloning PCR fragment into the expression vector

Mike Autry jma at ddt.biochem.umn.edu
Thu Mar 7 13:27:14 EST 2002


What about purity of agarose?  What brands and types work well w/ gel
purification and downstream ligation of DNA?  

These issues have impacted my work in the past.

-----Original Message-----
From: "Michael L. Sullivan" [mailto:mlsulliv at facstaff.wisc.edu]
Sent: Thursday, March 07, 2002 12:10 PM
To: methods at hgmp.mrc.ac.uk
Subject: Re: Cloning PCR fragment into the expression vector


I have to disagree with you here.  Virtually every ligation I've ever
done
(and we're talking about hundreds here) have involved a gel purification
step, and quite frankly, I rarely have a ligation fail.  I just don't
think
sulfate contamination in ligations is a practical problem-- in fact your
post is about the first time I've heard anybody mention it.  In my
opionion, the most important things are to make sure one is putting
appropriate amounts of DNA into a ligation reaction, and if one is gel
purifying a fragment, to make sure UV exposure of the fragments is
minimized.

Mike

>the agarose gel is the problem here.  Dont do all that. the PCR product
is
>clean enoughto do the ligation with.  Run it through an agarose gel and
you
>get sulphate contamination. However low sulphates are in the eluate,
ligases
>still show inhibition.  You can try shot gun cloning or use a
commercial kit
>for cleaning the PCR product.  you can try quiagen or invitrogen.  I
used to
>just take some of the PCR product and ligate  into the EcoRV site of my
>clone and get lots of clones.  Anyway, the cardinal rule is try to
avoid the
>agarose step as much as possible in any ligation work.  Remember
whenever
>you elute the DNA from an agarose gel, whatever you do, there will
always be
>traces of sulphates in the DNA obtained, which is enough to inhibit the
>ligase.  But blunt end ligation is quite easy when you use the PCR
product
>as such.

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

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