Blunt-end cloning

[Chi-kuang Wen]

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 
: appreciated

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).

Chi-Kuang