I would like to make primers for my qPCR. For that, I need a reliable software. I know the (ABI) Primer express software(paid) is preferred, but do you know any freeware that does the job well?
You can use the same rules for qPCR primer design as you do for end-point; just shoot for amplicons between 75-150bp if possible.
Also, make sure you validate your new primers after they arrive by performing a standard curve using template that is similar to your experimental samples to determine the dynamic range.
Any of these are nice:
Primer3 - http://primer3.sourceforge.net/
Primer-BLAST - http://www.ncbi.nlm.nih.gov/tools/primer-blast/
IDT PrimerQuest - http://www.idtdna.com/Scitools/Applications/Primerquest/
Primer Bank - http://pga.mgh.harvard.edu/primerbank/
RTPrimerDB - http://medgen.ugent.be/rtprimerdb
You can use the same rules for qPCR primer design as you do for end-point; just shoot for amplicons between 75-150bp if possible.
Also, make sure you validate your new primers after they arrive by performing a standard curve using template that is similar to your experimental samples to determine the dynamic range.
Any of these are nice:
Primer3 - http://primer3.sourceforge.net/
Primer-BLAST - http://www.ncbi.nlm.nih.gov/tools/primer-blast/
IDT PrimerQuest - http://www.idtdna.com/Scitools/Applications/Primerquest/
Primer Bank - http://pga.mgh.harvard.edu/primerbank/
RTPrimerDB - http://medgen.ugent.be/rtprimerdb
Thank you ver much for your nice response. I shall look for it!
Use the primer3. try to design the primer with 0 max 3' self complementarity and low value of max self complementarity. This will help you to get better reaction efficiency with least chances of secondary structure formation.
We use Primer Premier 5 (I like the feature in this program, which allows to manually edit and analyze the primers suggested by the machine). The catch is that the software is not free.
Another addition for manually designing primers: if working with eukaryotes - try and place the primers in exons that are separated by a (large) intron. Doing this in primer3 is a bit tedious, as there is no automation to this. NCBI primer designer can do that more readily, but I have not found a way to force it to generate primers that detect all splice variants of a gene. This way your small amplicon from cDNA will outperform any residual gDNA amplicons. We have very good experience with this (SYBR green based qPCR + TRIZOL or column based total RNA extraction, no DNAseI). Of course, you should validate your primers with -RT controls to evaluate gDNA amplification.
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