Problems with ligation after GeneCleaning
N.P.Dantuma at biol.ruu.nl
Thu Aug 22 03:08:35 EST 1996
Gabby Gerlitz <gabiger at zoot.tau.ac.il> wrote:
>> On Mon, 19 Aug 1996, Steven Goldberg wrote:
>> > We have been having a problem apparently due to GeneClean and I am
>> > wondering if others have been seeing it too. After PCR, we remove salts
>> > and clean up the fragment using phenol:CHCl3, precipitate, then perform a
>> > digest to cleave restriction sites on the end of the fragment. The
>> > reaction is electrophoresed on a TAE agrose gel and the fragment is cut
>> > out and purified using GeneClean. We have been getting good yields of the
>> > fragment, but it has been impossible to get it to ligate into the
>> > appropriately digested plasmid vector (which was also cleaned up with
>> > GeneClean). Since we are digesting with two different enzymes (such as
>> > EcoRI and XhoI), in theory the vast majority of transformants should
>> > contain a vector plus insert, but we only get some form of the vector
>> > alone.
>It seems that your PCR fragment was not cut by the restriction enzymes.
>Try clean the PCR fragment from the reaction mix with Quigen's kit for
>that perpose (instead of phenol etc.). Make sure that you use enough enzymes
>for the cutting (you need much more enzyme in relation to cutting plasmid)
>and cut for long time (~4 hr.).
>I had similar problem and got over it by those steps.
>Dep. of Cell Research and Immunology,
>Faculty of Life-Sciences,
>Tel-Aviv University, Israel.
>E-mail: gabiger at post.tau.ac.il
I agree with Gabi that the digestion may be the problem. I have no
experience with GeneClean (we use Qiagen) but maybe your 'cleaned'
products are containing something which may interfere with your
digestion. I think that in addition to the modification suggested by
Gabi I would also include controls (especially) when you cut your
vector. So include also the single digests with EcoRI and XhoI in the
same buffer which you use for the double digest and check those
digests on gel. Don't use to much units of EcoRI because of star
actvity and use for your double digest the buffer recommended for
Good luck ,
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