How to start a qRT PCR primer design?
Hi, I am trying to create a primer for a qRTPCR reaction but do not know where to start.
Answers
This site has some helpful information: http://www.premierbiosoft.com/tech_notes/PCR_Primer_Design.html
I have always found this site helpful http://www.idtdna.com/analyzer/Applications/OligoAnalyzer/
Make sure the melting temperature over 60 C and there are no hair pins.
They also sell primers for relativity cheap! :)
You can search for already existing primers at http://pga.mgh.harvard.edu/primerbank/ (Harvard primer Bank)
Only choose the primers that have been validated, these are sure to work!
I use Perl Primer http://perlprimer.sourceforge.net/
You can Blast the primer set to make sure it is not found somewhere else in genome. i usually use three diffrent primer sets for testing so that hopefully one of the three will work. Run these three primers with some control cDNA and check the melting curve, It should be a nice clean curve with only one peak unless you have hairpins or primer dimers





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