(none)

Mr. G. Anil Kumar mam at CGE.IISC.ERNET.IN
Sun Aug 31 04:39:32 EST 1997


Dear friends

I am having a problem in cloning a fragment. This is what I am doing. I
have already an 1.8kb insert in pBSK+ and is in reading frame. This clone
has a 420 bp C-terminal end missing. This clone is toxic to E.coli and
will not grow in IPTG plate. Now I am trying to ligate the rest of 420bp,
for that I have digested the pBSK+ clone with xhoI, dephosphorylated and
ligated with an 1.7kb XhoI insert. The control that is dephosphorylated
pBSK+clone is not giving any colony as expected on amp plates but I am
getting a lot of colonies in both IPTG plates and minus IPTG plates with
the ligated vector plus insert. Upon
analysis, I have found that about 29 of 30 has band migrating exactly at
3kb position (vector alone) and one had a 0.7kb insert and 4.2kb band.

Result expected : 1.7kb XhoI insert
                      plus
                 4.2kb band containing vector and Nterminal part of clone

Result obtained : 29/30 contain 3kb band only
                    one contain 4.2kb plus 0.7 kb insert

I am really in trouble. Insert alone also I have put for ligation and has
got any colony suggesting no contamination

My question.
If dephosphorylation is not worked, at least I should only get the
religated 4.2kb instead of what I am getting here 3kb. As the insert is
highly toxic to E.coli, it it that somekind of deletion of the insert
or throwing of insert taking place? But my worry is that being not a
directional cloning I must get some clones which is ligated in opposite
orientation and that is not toxic to E.coli. Why am I not getting that
kind of clones?

Please enlighten me with your experiences

ANILKUMAR








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