I am using a mixture of 2 forward primers and 4 reverse primers in a single pcr-mix for the detection of Arbuscular Mycorrhiza Fungi and I was wondering in what concentration should I use them. The reference article I am using is Kruger et al. (2009) where they describe the mixture of 2-4 primers respectively as "one primer" and the concentration they use of this "each primer" is 0.5 uM. I doubt whether the concentration of the 2 or 4 primers together would be 0.5 uM or each single primer's concentration should be 0.5 uM.
Hi there,
Primer concentration is meant to be applied to each primer whatever the PCR application. But in the peculiar situation where forward primers are less than reverse primers (or the opposite situation), it could be worth taking into account than forward primers will be consumed faster than reverse primers during the PCR and therefore either increase the concentration of forward primers or decrease the one of reverse primers.
Dear,
I think the mentioned 0.5uM concentration is for each primer (final concentration in the reaction mixture). I usually use 0.5uM final concentration of each primer (i.e. from a 10uM stocks of the primers I use 1uL for each) for a 20uL volume of reaction mixture and it works nice.
Good luck.
When preparing multiplex PCR reactions you can be obliged to test different proportions of the different couples of primers. Besides the final concentration, the relation among different couples must be tested, starting by 1:1 and followig by 1.5:1 then 2:1 and even 3:1. The higher the number of PCR reactions you are multiplexing, the more important the proportion testing. Good luck!
Testing of different concentration is the key, as Luis mentioned before! Important role is also playing the ''efficiency'' (or quality if you prefer), influencing the necessary concentration. Be systematic and methodical and you will find the perfect primer mixture !
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