Insert seems to stimulate self ligation of pGEX vector using EcoRI single digest method

Filippo Geuna filippo.geuna at unimi.it
Mon Aug 26 12:43:52 EST 2002


Hans Wessels <hansw at sci.kun.nl> wrote:

Dear Hans,

the overall setup of your experiment is quite straightforward and I
cannot figure out weak steps. According to my experience and that of
some colleagues of mine, I have noticed that the CIAP enzyme often
generates improper constructs (maybe for the presence of residual
contaminating unspecific nucleases). I prefer working with Shrimp
Alkaline Phosphatase (SAP), which is also sensitive to moderate heat for
inactivation purposes. Another trick that I sometimes use when cloning
apparently difficult products is to check the cut vector after
restriction enzyme and SAP treatment by self-ligation.
Basically I cut the vector, treat it with SAP, self ligate it with T4
ligase and load the product on a preparative gel. I recover the slice of
gel that contains the UN-religated portion of the molecule (linear) and
use it for downstream cloning of my product of interest. It usually
works.
Hope this can help.
Sincerely,

Filippo Geuna


P.S. Another question: have you tested the complete digestion with EcoRI
of the PCR product? Is the EcoRI site designed sufficiently recessed
with respect to the end of the amplification product?



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