Amplification from a single colony, any thoughts?

Airlie Sattler airlie at mindspring.com
Fri Feb 7 10:27:46 EST 1997


Dear Molbio.evolution readers,

Our lab has successfully amplified the ITS1 and ITS2 rDNA region in the
group of algae we are studying using DNA from a quick and dirty CTAB prep
as the template. I am now attempting to modify our protocol to amplify the
region using a single colony of algae dropped into the reaction mix as our
template. In my first attempt I had no amplification at all. I extended
the number of cycles and the extension time and got a very weak, fuzzy
band from one of my tubes. (My control was negative). I'm going to
continue to play with this, but I thought I'd throw it out onto the net to
see if anyone had any suggestions. Basically I need to get stronger
amplification and a sharper band. Have any of you had success in
optimizing a PCR protocol like this? Anyone have any tips for amplifying
from a single colony (about 10-70 cells)? At the moment we are using a
bare bones reaction mix with no additives other than BSA. Do you think
something like DMSO would help?

Please send any thoughts you might have to: asattler at beta.loyno.edu
Thanks in advance for any help.

Sincerely,
Airlie Sattler



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