Pcr primers - (Feb/04/2012 )
I have to do a multiplex pcr wherein I have to add a total of 6 primer pairs to the same reaction tube.
I need to know whether I can add all primers togather and make cocktail and store them so that whenever needed i can take out that much quantity and add to the reaction mix
as otherwise this step will consume lot of time and there are chances of mistakes
Let me know what are the factors i shd consider before making a cocktail
I think either way should be OK once you have optimized the amount for each pair in the reaction.
Sometimes primers can anneal to others, especially when there are 6 pairs of them. Generaly each pair is designed to be not-complementary so you should at least try to put a pair in one tube, that will cut the number needed to half.
Then you need to optimise amount of each pair that will work together as pcrman said. They you can try to mix that amounts together and see if it works upon freezing and thawing.
If you encounter problems with the mixed one, but not separates, you can heat and quickly cool the mixture before adding it to the reaction, that should disrupt the secondary structure.