Should my forward primer volume in the mastermix should be same with other two reverse primers? or do I need to double the volume of forward primer so that the sum of volume of reverse primer 1 and 2 equals forward primer?
use the usual amount of forward primer as for single primers pcr. The primers are in huge excess and the forward primer amount will cope easily with 2 reverse primers. Having too much primer just increases the likelihood of primer dimer which will make your pcr less efficient
use the usual amount of forward primer as for single primers pcr. The primers are in huge excess and the forward primer amount will cope easily with 2 reverse primers. Having too much primer just increases the likelihood of primer dimer which will make your pcr less efficient
Hi I agree with Dr. Rutland and Dr. Suleman.
Primers are in excess in the assay mix. Do a regular PCR assay first, see the results carefully and plan the next step.
In agreement with others, for F primer, the same volume with other two reverse primers would be enough.
Good luck
While you have a good answer on how much primer to use, I'll add that you'll need a FULL set of controls. In general, the smaller of your two PCR products will be amplified more than the larger product. You might want to consider re-testing any that do not show the larger product with a single primer pair just to rule out amplification bias.
Thank you very much for all your kind answers! it helped me clarify my question!
In agreement with others, for F primer, the same volume with other two reverse primers would be enough.
Good luck
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