cloning pcr from VENT,Pfu etc.
rogerM at qimr.edu.au
rogerM at qimr.edu.au
Mon Nov 8 00:32:48 EST 1993
Dear clonetters,
Does anyone have experience cloning pcr fragments from the other
thermostable polymerases particularly VENT and DEEPVENT from NEB
and PfuI from Stratagene. What successes and/or failures have people
experiences. Also has anyone had problems cloning pcr fragments
using degenerate primers containing Inosines. We have had one
success with these primers but other different degenerate primers
have been totally unsuccessful. There are white colonies showing
up but the majority show up to be deletions if >100bp in the insert
and unsequencable due to apparent deletions in the polylinker. The
fragments were tested against control fragments from specific pcr
primers by self-ligation and the test set totally failed whilst the
controls all self ligated.Any tips, suggestions or spelling flames
are welcome :-). As a final note any tips to reduce my golf handicap
from seasoned pcr golfers welcome?!!!
Roger Mitchell.
e-mail: rogerM at qimr.edu.au
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