Three fragment ligation

Pamela Norton pnorton at
Fri Jan 17 12:24:22 EST 1997

In article <32DF3CC3.5FE8 at>, Andrew Doherty
<A.Doherty at> 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.


     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

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