I want to do qPCR of multiple genes but the problem is that the anealing temperature of multiple primer sets varies a little. How can i address this?
Hi Junaid,
You can try to combine annealing and extension (about 60C) steps, in my experience also using master mix which already contains an optimal concentration of MgCl2, which works with nearly all primer combinations is helpful for optimising qPCR reactions.
It depends on how large the difference in the annealing temperatures is. If your differences are very small (
Hi,
I have amplified samples with different annealing temperatures in the same run using (Gradient PCR). This allows you to set maximum and minimum annealing temperatures and each lane will have intermediate temp between those minimum and maximum. It is mainly used to check the optimum annealing temperature for primers.
https://www.drugdiscoveryonline.com/doc/using-gradient-pcr-to-determine-the-optimum-a-0002
Thanks to all your valuable answers. I have I am going to buy a sybergreen PCR master Mix. The temperature differences are not known yet i have to optimize them. For this I have planned to do gradient PCR. But to optimize 60 gene primers is a big target, it will take much time, and resources also. I want to to address this also.
Do you need to run in a routinely way different set of primers with differences up to 5°C at the same time? If you do not have a PCR thermocycler to dispose more than one temperature, my suggestion is that you design your primers with an identical short tail, of 10-16 nucleotides in length, for all your primers a tail for F and other tail for R primers.
Dear Junaid Kori
I had had this problem but at small scale of samples. Also I had a short well designed primer and I use master mix already in amplification. However, I over come this problem by trying gradient PCR with different annealing temp. (from the min. to Max.). All the best
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