In article <MAILQUEUE-101.930804101416.334 at micro.uct.ac.za>,
ED at micro.uct.ac.za ("Ed Rybicki") writes:
|> If you don't want to use Taq to T-
|>tail, or to T-tail with terminal transferase and ddTTP, then you could
|>what someone here has mooted, which is to cut a plasmid with any
|>that gives a T at the end of a 3'-overhang, then fill in with Klenow
|>dC-, dG- and dTTPs (note: no dATP) to get (eg.):
|>|> vector PCR amplimer vector
|> 5'-XXXXXXXXXXXXXXXXXGACT XXXXXXXXXXXXXXXXXXXXXXA GTCXXXXX-3'
|> 3'-XXXXXXXXXXXXXXXXXCTG AXXXXXXXXXXXXXXXXXXXXXX TCAGXXXXX-5'
Unfortunately if the end has a 3' overhang it can't be filled in with
Klenow, which requires a 3' recessed end as template/primer. The T-tailing
with Taq has worked OK for me, but the background of colonies with empty
vector was so high that I routinely used colony lifts to find the plasmids
with inserts. I am not really satisfied with any of the methods I've tried
for cloning PCR products; it always seems to be more difficult than cloning
comparably-sized blunt ended restriction fragments.
Research Assistant Professor
Forest Biotechnology Group
North Carolina State University
Raleigh, North Carolina 27695-8008 USA
telephone or fax (919)515-7801
e-mail rosswhet at unity.ncsu.edu