Useful bookmarks
for probe and primer design:
Use free
online Tm calculators to see what the Tm for primer and probe sequences are.
We use the Operon calculator, as it also has a good tool to check possible
primer-dimerization sequences. Reach it from www.operon.com then
select DNA Synthesis, then select Oligo Toolkit.
This is a
link to Primer3 software. It is software that allows for primer design and
also helps picking an internal oligo sequence to these primers (a probe
sequence). Like most primer design algorithms, it has the disadvantage
of not taking into account secondary structure issues that are paramount in primer/probe
design for real-time PCR. With that caveat in mind, it is a good place to
start the design process, if you are not inclined to do it by eye
(i.e. scanning the sequence yourself). Then you can check your sequences at
the folding site (described below).
This is the
site for the Basic Local Alignment Search Tool from the National Center for Biotechnology Information. Use
this site for checking specificity of probe and primer sequences.