PCR cloning in pDK101 XcmI, reference

[Andy Law Big Nose]

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

( Andy.Law @ bbsrc.ac.uk )
( Big Nose in Edinburgh )