Flumoxing ligation problem

Steven Goldberg goldberg at bms.com
Thu Jan 2 13:33:51 EST 1997


In article <32B7B7F1.4CED at Bris.ac.uk>, Andrew Doherty
<A.Doherty at Bris.ac.uk> wrote:

> HELP!!!!!!!!!!
> 
> Any ideas will be most welcome
> 
> Yours confusedly
> Andy D
> -- 
> *************************************************************
> Dr Andrew Doherty               email -  a.doherty at bris.ac.uk
> Dept. Anatomy                   Tel (0117)9287421
> School of Medical Sciences      Fax (0117)9287402
> University of Bristol
> University Walk
> Bristol UK
> BS8 1TD
> *************************************************************

ANDY:  I have occasionally have had severe problems ligating PCR products
into plasmids.  Often it is due to poor digestion of the PCR molecule I've
synthesized.  Some restriction enzymes require a lot of extra bases before
the restriction site (see New England Biolabs catalogue), while others
need an additive like BSA for good activity...you should double check with
your enzymes.  Also, I've given up on GeneClean for gel purification since
I have been having a lot of problems with ligations after using it. 

In a recent case, I could not get a PCR fragment with NdeI and BamHI sites
to ligate to an expression vector with the same sites.  I was ending up
with transformants containing vector alone despite gel purification after
endonuclease cleavage.  I tried several changes which in combination gave
me the desired vector + insert:

1.  I added an extra site to my 5'-end primer so that it contained an
EcoRI site upstream of the NdeI site

2.  After PCR, I extracted the reaction with phenol:CHCl3 and precipitated
with EtOH.  I digested the product with EcoRI and BamHI and cloned into
pZeroI at identical sites.

3. Then I removed the PCR fragment from pZeroI using NdeI + BamHI.  In
this digest I included acetylated BSA since it is supposed to enhance NdeI
activity (this is supposed to be a very finicky enzyme).  At the same time
I digested my expression vector under the same conditions.  The reaction
was left overnight at 37oC.

4.  I purified the desired fragments using the QIAquick Gel Extraction kit
and ligated, then transformed.  Ca. 50% of my transformants contained the
correct insert.

Obviously you might have to make changes for your particular case, but the
above procedure should help you to get the construct you are looking for.

Good luck.

Steve



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