I'm using NCBI's Primer-Blast to design primers for qPCR. When I get the results, is the first primer pair listed generally my best bet? I ask because I recently noticed that the first primer pair result for rat PPIA has several more potential off-target amplicons, but has a lower 3'-complementarity score (2, versus 3 for the second pair).
Dear Anne Tye,
I was using NCBI’s Primer-Blast until I discovered more precise programs that you can download for free. I propose you to try AmplifX or PerlPrimer software.
Best regards
Ait Lahmidi Nassima
Hi Anne,
You can go for Primer3. It is decently reliable.
http://primer3.ut.ee/
I have had good luck using the IDT qPCR primer design tool: https://www.idtdna.com/Primerquest/Home/Index
Primer3 (http://bioinfo.ut.ee/primer3-0.4.0/) is quite good when you come you design your own primer, find your sequence and paste it on there like this Exon1[]Exon2 or exon 2-3 or 3-4. You get the idea, just put the square brackets between the two exon sequences.
For an even easier choice, I use Primer Bank (http://pga.mgh.harvard.edu/primerbank/). Some of the primers on there are already undergo optimisation and you can see the end product on the gel photo they provided. However, in general I found their product size are slightly larger than my ideal range. You can always give it a go though.
Hope it has been helpful, good luck.
Primer3 is very good. But a simple alternative would be to buy predesigned kicqstart sybr green primers from Sigma Aldrich. They are not very expensive and work very well in most cases.
http://www.sigmaaldrich.com/catalog/product/sigma/kspq12012?lang=de®ion=DE
I quite like Primer3 but usually set the temperatures a bit higher than default
Hi
Primer 3 is reliable tool for primer design, also primer blast in NCBI, and Vizprimer software you can use to even SNP detection, also many online program available to design primer
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