I'm designing some primers for qRTPCR. Does anyone have any suggestions for a good qRTPCR primer design tool? (I expect to need to check for hairpins and dimers and what not, but would prefer not to do everything by hand....)
The only issue I had with this tool was that it chokes on its auto-gene-lookup function, and it needs introns in lower case with exons in upper case to work properly. I wrote a little script however to format a gene sequence, and you're welcome to use it.
http://naus-lab.com/tools/intron_exon.php
Just get the genomic genbank file for your gene of interest, and copy-paste the sequence into the left hand text box (numbers are fine to include, but nothing else), then locate the mRNA exons in the genbank Features list (should look like: "mRNA join(1..348,2472..2614,4983..5197,7510..9321)"). Copy-paste ONLY the list of numbers (i.e., "1..348,2472..2614,4983..5197,7510..9321") into the right hand text box, and hit 'Run Sequence'. You can copy-paste the output directly into the lower IDT gene entry text box, and click 'Design Assays' to get your primers. It will include a probe sequence as well, which you won't need if you're using SYBR Green, but you can just ignore that and grab the fwd-rev primers.
Hope this helps, and feel free to get in touch if my little web tool is sufficiently non-user friendly ;) (I can always make it better if others start using it)
To design primers on multiple alignments, I normally use Primaclade (https://131.204.120.103/srsantos/primaclade/primaclade.cgi) which is very unknown but very user-friendly and easy-going. When I design primers on a known sequence I just use Primer-Blast (http://www.ncbi.nlm.nih.gov/tools/primer-blast/). Here, you should edit something: advanced parameters > Primer parameters > Table of thermodynamic parameters - change SantaLucia 1998 into Breslauer et al. 1986. This gives you annealing temperatures more "real" for the standard conditions we normally work with. Primer-Blast gives you also info on dimer formation.
To deepen dimer and hairpin formation risk, I totally recommend the use of OligoAnalyzer (http://eu.idtdna.com/analyzer/applications/oligoanalyzer/).
You get detailed information about the risky deltaGs, too.