PCR cloning in pDK101 XcmI, reference
Andy Law Big Nose
Andy.Law at bbsrc.ac.uk
Fri Aug 18 04:41:58 EST 1995
In article <40vben$t2m at hobbes.cc.uga.edu>, russell at dogwood.botany.uga.edu wrote:
> "Direct Cloning of DNA Fragments Generated by PCR"
> Kovalic, D., Weisblum, B.
> 1995, Methods in Neurosciences, volume 26:255-261
> Sarkar, G., ed., Academic Press
> I learned of this review article containing suggested protocols for use
> of the vector pDK101 with the enzyme XcmI to clone PCR fragments,
> and thought I would pass the reference along.
> Some notes from the article:
> - pDK101 is available from ATCC in strain number 77406
> - pDK101 has XcmI sites that leave 3' unpaired Ts on the vector,
> following removal of a 16 base pair stuffer fragment.
> - they recommend gel purification of the vector away from the 16 bp fragment
> - they recommend the addition of a kinase step just before adding ligase
When I tried to use this plasmid, I found that the blue colonies that you
inevitably get were very very pale blue. Be very careful when you select
colonies that you are genuinely selecting whites.
( Andy.Law @ bbsrc.ac.uk )
( Big Nose in Edinburgh )
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