2 replies to this topic

[murrayk91884]

I'd like to know how to go about vaildating my real time primers before I actually begin to use them on real samples. Any suggestions?

[wuxx0153]

murrayk91884, on Apr 22 2009, 07:35 AM, said:

I'd like to know how to go about vaildating my real time primers before I actually begin to use them on real samples. Any suggestions?

This is what we do when we have new real-time PCR primers:

Run PCR reaction with new primer in regular cycler with identical program set up as real-time PCR. Check PCR product on gel and make sure only one product can be identify. Sequence it to make sure this is the amplification fragment we have designed to get.
After product confirmed, we begin primer titration to evaluate PCR efficiency, and optimize if necessary.

People say we are too picky, maybe so; but I think you should at least run titration to evaluate primer efficiency and pay attention to disassociation curve to ensure there is only one product.

[pcrman]

Quote

People say we are too picky

Not at all. You are doing real science.