Amplification from a single algal colony, any thoughts?

Airlie Sattler airlie at mindspring.com
Thu Feb 13 08:59:12 EST 1997


Dear molbio.methds-reagnts 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 eukaryotic cells)? Even though our
target DNA is present in multiple copies in the genome, I'm concerned that
there might be too little DNA there to start with. PCR is well known for
being able to amplify from next to nothing, but how do you optimize a
reaction to do that? 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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