Redilute Primers
Primer were originally diluted by adding 1 µl of each primary forward and reverse primer at a
primer stock concentration of 100 pmole/µl (100 µM) into 78 µl of sterile distilled water to give
a diluted primer stock concentration of 1.25 µM for each primer. Approximately 4% of the time,
the original nested PCR was not successful. When this occurred, the primer stocks
corresponding to the ~4% of ORFs where the PCR failed were rediluted by adding 1 µl of each
primary forward and reverse primer at a primer stock concentration of 100 pmole/µl (100 µM)
into the 79 µl of remaining diluted primer stock, bringing the final concentration to
approximately 2.5 µM for each primer and the standard nested PCR reaction was repeated.
Using these rediluted and more highly concentrated primers in a second set of PCR
reactions was successful in producing a PCR fragment of the expected size about 86% of time.
Approximately 50 ORFs needed additional attention in order to get the PCR fragments to
amplify. In some cases success was achieved after new ORF-specific primers were ordered that
were six bases longer than those used in the initial PCR reaction. Sometimes, especially for long
ORFs, the elongation time used in the PCR protocol was increased to 8 min.
The success with longer primers could be due to errors in the original primer synthesis. It
is also possible that differences between the template and published sequence were causing some
of our difficulties, since the ~260,000 bases in primer sequences is large compared to the target
sequence quality of less than 1/10,000 errors per base.