Three fragment ligation

Andrew Doherty A.Doherty at
Fri Jan 17 03:48:03 EST 1997

Hi everyone

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.

Ta in advance
Andy D
Dr Andrew Doherty		email -  a.doherty at
Dept. Anatomy			Tel (0117)9287421
School of Medical Sciences	Fax (0117)9287402
University of Bristol
University Walk
Bristol UK

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