ckwen at expert.cc.purdue.edu
Fri Jun 14 11:26:10 EST 1996
John Donald (jdonald at deakin.edu.au) wrote:
: I have been using Stratagenes pCRscript blunt end cloning kit with
: limited success. I often get many blue colonies and as a rule the
: white colonies are not recombinants. The PCR products are polished
: prior to cloning and are added to the cloning reaction at 40-100:1
: insert to vector. It seems to me the Srf1 enzyme is not cutting the
: plasmid that efficiently and therefore the vector is somehow
: preferentially ligating on itself. I have read that blunt end
: cutters can delete nucleotides. Is this true. Any advice would be
It seems to me that you might use too much inserts in your ligation reaction.
Besides, you mentioned a 40-100:1 ratio of insert:vector, is it based on
mass(ug) or on molecule number. Either way, even you polished the inserts< I
Assume you use T4 DNA polymerase>, there are still heterologous DNA which can
not be processed, and under the condition of high ratio of insert: vector, those heterologous DNAs may compete with the vector and generate plasmid:heterologous DNA which can not be functioning after transformation.
Usually, I prepared EcoRV cut plasmid and phastase it. Use 1:1 ratio of insert
:vector (in molecule number ratio) for an overnight ligation. Do not use too
much ATP which certainly inhibit the blunt-end ligation. The transformation
is very good to me ( I did not count, just get lots of white colonies).
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