problem cloning 2.5 kb fragment - (Mar/30/2006 )
I am trying to clone a 2.5kb gene(cDNA) which is in
pGEMT-EZ vector. The gene can be released from this
vector only using NotI enzyme (the other enzymes cut
the gene internally). I've repeatedly tried to clone
this gene into the NotI site of PCRII-Topo vector (4kb)
using different ligation ratios and buffers,with and
without PEG etc.,I've also tried treating the vector
with CIP, but then no colonies come. if i do without
CIP, i get a fair amount of vector religations(both
blue and white colonies). control ligations with uncut
plasmid and vector alone religated work very well.I also
tried amplifying the gene from pGEMT using a proofreading enzyme and
attempted to put it directly into a 12kb vector,
but was'nt successful.i also find it impossible to
clone the same gene amplified full length using advantage
proof reading enzyme back into pgemt-EZ.
i have this problem only with this particular gene. other
genes(upto 2kb size) have been successfully cloned.
kindly help me solve this problem.
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i heard that when u have DIFFICULt genes to clone, some people use LOW MELTING AGAROSE instead of normal one, and try using SOC medium instead of LB medium (if u use LB). or added some glucose to LB.
maybe this will help.
good luck
pGEMT-EZ vector. The gene can be released from this
vector only using NotI enzyme (the other enzymes cut
the gene internally). I've repeatedly tried to clone
this gene into the NotI site of PCRII-Topo vector (4kb)
using different ligation ratios and buffers,with and
without PEG etc.,I've also tried treating the vector
with CIP, but then no colonies come. if i do without
CIP, i get a fair amount of vector religations(both
blue and white colonies). control ligations with uncut
plasmid and vector alone religated work very well.I also
tried amplifying the gene from pGEMT using a proofreading enzyme and
attempted to put it directly into a 12kb vector,
but was'nt successful.i also find it impossible to
clone the same gene amplified full length using advantage
proof reading enzyme back into pgemt-EZ.
i have this problem only with this particular gene. other
genes(upto 2kb size) have been successfully cloned.
kindly help me solve this problem.
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hello Im0000,
Your insert isn't *huge* so should clone okay - and it can obviously be propagated in pGEM... Presumably you want to subclone into a TOPO vector to make a transcript or something? If this is the case, you can re-amplifying from your pGEM clone using a primer which includes a T7 promoter site, if all else fails...
It's most likely to be a transformation problem, due to the size of the construct. Have you tried using electroporation, with high-efficiency competent cells?
as for the problem cloning the re-amplified gene back into pGEM: the advantage polymerase might leave 3'-A overhangs which won't ligate into pGEM-EZ. I think this is a polymerase mix, and may not give blunt ends - check with the manufacturer (Clontech?).
D.