Major Ligation Trouble - CIAP again?

Andrew Doherty A.Doherty at Bris.ac.uk
Fri Jan 24 04:05:29 EST 1997


Hi Everyone,

I've got a headache - brought on by trying to ligate two (or three)
fragments together. Basically, they don't want to know about it. I've
just looked at my lastest attempt and after pulling my hair out, have
turned to you. The problem is this. I'm TRYING to make some GFP fusion
constructs. In order to make a full length fusion, I've modified the 3'
end of my receptor (GluR1) by PCR, and that went fine. I'm now trying to
subclone this modified receptor to an expression vector using EcoRI/XhoI
directional cloning. The Vector (pcDNA1/amp from Invitrogen) is rescued
from another plasmid (to ensure that both sites have been cut),
de-phosphorylated using CIAP, gel purified and used in ligation with
BM's T4 ligase. On a gel the ligation looked fine - nothing with no
insert control, a ladder of products with the insert present. I've then
transformed by electrporation using the ligation mix as it is (test
ligation and control), a sample of normal GluR1 in pcDNA1/amp as a
positive control, and DNA extracted from the ligation mix. I get
thousands of colonies from my positive control, and nothing from
anything else - HELP!!!!!!

I've just been throught the archives of this group, because I know that
the problems of CIAP have been extensively discussed, most of that
discussion has centered on inactivating CIAP by heating. My question is
really, is it possible that CIAP can cause problems following gel
purification or does it contain exonuclease activity which could degrade
the DNA? or should I simply move over to SAP?

Thanks for your help

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
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