When designing primers in Gap4, I pay attention to the output in the
message window. It tells you the GC content, Tm, and size of the oligo
that has just been picked. It also gives you a score. Recently, I've
been using the oligos at the end of the list because they have the highest
score. However, many of these reactions using these primers off M13 DNA have
not been working. I know the sequence in the region is good. I'm pretty
confident in the quality of the primers being made and I know our
sequencing reagents are good--including the DNA. Should I not be using
the oligos with the highest scores? Most of these primers have GC > 40%,
Tms between 50C and 55C, and are 17-20bases in length. I'm using the
recommended cyling profile from ABI for Big Dye Terms which works great
with M13 DNA and the -21 primer. Thanks.
--
Paul Shinn
Sequencing Coordinator ,___o
pshinn at neomorph.bio.upenn.edu _-\_<,
Arabidopsis thaliana Genome Center (*)/'(*)
http://genome.bio.upenn.edu/ATGCUP.html
(215) 573-7256
