PCR blunt end cloning

HIDEW at SIMSC.BITNET HIDEW at SIMSC.BITNET
Fri Oct 18 09:45:00 EST 1991


WRT the message posted about PCR product blunt-end cloning by
Hirdeypal S. Bhathal:

He inquired as to the effacy of his blunt end system, and noted that
he was getting "false" white colonies after subcloning.



I KNOW the story!!!


        Here I am also trying to do a great deal of blunt end cloning,
and frankly hate it. But there is a method that DOES (I promise) yeild
some results, even in the hands of a clumsy scientist such as myself.


I have invested in an Invitrogen T/A cloning kit. The principle of this
kit is to utilise the ragged end of the PCR product that has an extra A
overhang incorporated by the action of the thermostable polymerase.


The vector for this system has a (I think) HincII digested site that
results in a matching overhang/end.

The cloning frequency is good for the system because the kit is supplied with
*VERY* high competency cells, reagents (including ligase and optimised buffer)
and control elements.

It is the only way I have managed to subclone my more reluctant blunt-ended
PCR products. I am in no way connected to the copany that makes this thing,
but feel it's OK to say it works, and works WELL.


I hope that this will be of some use. I feel it pertinent to add that
there is a lot of regular blunt-ended cloning done in this lab,
with extremely variable results.


Win Hide
Lab of Molecular Systematics
Smithsonian Institution


(some people have nucleotide fingers....some have plain 'ol paws...)



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