Primer problems?

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KatarinaLy
KatarinaLy's picture
Primer problems?

Hi!
I am having trouble with my qPCR. The housekeeping gene is nicely ampifyed but there is nothing happening with my primers. I have used Primer3 to design three pairs but non of them seem to work?
I have tryed different concentrations and different annealing temp but still nothing. So there is probably someting wrong with my design... Is there a better program for qPCR primers?

Katarina

Ckis
Ckis's picture
Primer3 may not give you

Primer3 may not give you optimal primers specific for qPCR.

I would use the following links for a low cost primer source. For each Gene symbol in the databank, you can get 3 different primers to try. We have ordered hundreds and the success rate is about 90% meaning we have to re-rder and try another primer for about 10% that we get. Once you get a generated sequence, just order from IDT tech...about $14 a pair on average....and make sure you get lab ready. Good quality from them.

This one is for human genes

http://primerdepot.nci.nih.gov/

This one is for mouse genes

http://mouseprimerdepot.nci.nih.gov/

A little more expensive but even better....go to this site below and order your primer from them. They will design and even validate your primer set for no more than $40 a pair. $20 if they already have it on hand. Whatever they send is guaranteed to work. We have ordered over a hundred from these guys and they do great work.

http://www.realtimeprimers.com