Greetings, I am interested in if anyone has done PCR on Chlamy
chloroplast DNA using the enzyme Pfu polymerase (cloned). I have used 3
different mini-preps for chlamy total DNA, and have tried annealing
temp's at 50, 52, and 55, DMSO at 2% or 8%, and cleaned out all of the
EDTA so the Mg concentration would be at the specs for the enzyme. I
have yet to generate the expected band.
The primers I use have a Tm of 65 and 56, and I have successfully used
these primers to generate site specific mutations in the psbA gene of
chlamy that we have cloned into E coli for genetic work. I can't seem to
get these primers to work on the total DNA extract from Chlamy.
Other items I have thought of is the length of time for annealing,
currently at 90 sec or 2 min, or the extention time, currently at 2 min,
with a final extention of 20 min at 72 C.
>From the mini-preps I usually reppt the DNA after the extraction with
isoproponal and wash with ethanol and let the EtOH evaporate for 30 min
or so. I haven't had any success with CC-124, ac-u-e (W.t, chlamy) or
the strains pBA157 (intron free copy of psbA in ac-u-e host) or the
possibly site specific mutant of pBA157. In other words, no luck across
the board.
Anyone have any suggestions? I am definately interested in amy mini-prep
protocol that people have used to successfully prep chlamy DNA as a
template for PCR, or any suggestions on the PCR conditions that have
been used. I can be reached at padden at ahab.life.uiuc.edu.
Thanks!
Sean Padden
