Three fragment ligation

Pamela Norton pnorton at lac.jci.tju.edu
Fri Jan 17 12:24:22 EST 1997


In article <32DF3CC3.5FE8 at Bris.ac.uk>, Andrew Doherty
<A.Doherty at Bris.ac.uk> wrote:

> There must be lots of people out there ligating three bits of DNA
> together instead of just two - but I'm having major problems with it.
> I'm constructing a fusion protein, but have very few restriction sites
> to choose from which I can use, hence the need for the three fragments.
> My vector is pcDNA1/amp (dephosphroylated) cut with NotI/SphI, my
> receptor cut with NotI/HindIII and GFP cut with HindIII/SphI. So putting
> all three together should give me a circular plasmid. The ligation looks
> fine on a gel - the GFP (800bp) and receptor (3Kb) both disappear and I
> get a ladder of products. But no colonies!! I know that the bugs are
> fine, as other transformations are working well - so my question is, are
> there any tricks in getting this sort of ligation to work or is it just
> luck??!!@# I'm using BM's T4 ligase at 4 deg C overnight.
                                         ^^^^^

Andy,

     Why 4 deg C? Or is this a typo? Ligations are commonly carried out at 
_14_ deg C. Room temp seems to work fine most of the time, and I believe
that it is preferable for blunt end cloning.

     Despite my questions, the temp doesn't seem to be your problem if you
see evidence of ligation. Sounds like a problem with your vector piece, try
preparing it without the dephosphorylation step, just gel purify it. 

     Good luck,

          Pam Norton

-- 
Pamela A. Norton, Ph.D.          Associate Professor of Medicine
Thomas Jefferson University
Philadelphia, PA 19107           p_norton at lac.jci.tju.edu



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