Cloning PCR fragment into the expression vector

[Michael L. Sullivan]

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
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