Help - cloning into dephosphorylated plasmid vector

Nick dos Remedios N.dosRemedios at uts.edu.au
Tue May 23 18:35:16 EST 1995


In a nutshell: I am unable to ligate foreign DNA into the ECORI site of
my plasid vector (pcDNAI) after the vector cut with EcoRI then treated
with C.I. alkaline phosphatase. Cut vector re-ligates back together quite
efficiently, therefore the problem seems to be the CIAP step but I have
been unable to fix it [I *think* my overhangs are being eaten by the
CIAP].

The result I get after ligating (O/N at 16 degC) a test insert (EcoRI
ends) of 1.7 kb and transforming into my bugs (MC1061/p3) is that I get
background numbers of colonies for all ligation reactions (background =
no insert control ligation reaction). i.e. trying 3 different molar
ratios of vector to insert (3:1, 1:1, 1:3).

CIAP treatment: I am using Promega CIAP molecular grade and have been
following their protocol. I have tried 2 different batches of this
enzyme. Cleaning up after the CIAP reaction seems to be critical and I
have tried the Promega method and both of the Sambrook et al. methods
(heat denaturing enzyme & digesting with proteinase K; followed by
phenol/chloroform extraction etc.). I have also tried heat denaturing
followed by GeneCleanII DNA clean-up and purification. NONE of these
measures have made any difference!

The only suggestion I have received from someone who has used this
vector, is, forget the CIAP step (i.e. don't dephosphorylate vector) or
else clone into two separate R.E. sites (directionally clone). However, I
already have cDNA adapted with EcoRI so I don't want to waste it.

If you have any suggestions please reply/post, I am desperate!

Nick

Nick dos Remedios                                Immunobiology Unit, UTS.
N.dosRemedios at uts.edu.au                         tel (02) 330 4045



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