I have two primers sets and DNA of 50 isolates of a fungus in which 34 isolates can amplify with the first primer and the remaining 26 with the second primer. However, I want to produce bands with all 50 isolates in a single PCR reaction. Annealing temperature for both the primers is the same.
As what I could understand out of your question is that your looking for PCR amplification using two sets of primers having similar annealing temperature in a single thermal program. So, the answer to this is yes you can do that. Prepare your PCR reactions according to the primer sets (like use first primer set in reaction mix of 34 isolates and second primer set in the reaction mix of remaining 26 isolates). Put you PCR tubes (all 34+26) in PCR machine and run it in the thermal program. You will get good amplification if primers have same annealing temperature. The other thing which you have to consider is PCR product size, if both primer sets are giving some what similar product size then you will get good amplification. But if very different then you have to adjust the time for denaturation, annealing and extension. Hope it will work for you and you will get you desired amplification.
I think Chhattar Pal is asking about using 2 or multiple primer used in a single Mastermix. yes It is Possible in fact theis specific type of amplication is called as "Multiplex PCR" see details on web.
Hey Chhattar, it is possible both ways as Parveen or Haris suggested you. there are other factors that you need to consider like how specific your primer pairs are, there may be cross hybridization, so you need to check couple of reactions before putting up all the reactions and certainly controls will be very important using single primer pairs. if the gene sequence that you are amplifying doest not differ much in these two type of isolates, you there is a possibility of amplicons from 5` primer from one pair and 3` pair of other. if primers are good, you are good to go . . . all the best
Thanks
i actually asked multiplexing of pcr primers
i have tried four primers in multiplexing pcr
but there was no amplification
what possible error may ocurred in that case?
Please anybody suggest me
I guess you checked primer pair individually and they are working fine. well if there is no amplification i would check for the PCR conditions and components. if PCR is working fine and Primes are also working fine separately, you should get something during multiplexing. check for the various PCR reaction components if something is gone bad. once you get something it may be unspecific, shorter than the expected, longer than expected, very low amplifications, some primers working others are not, etc. then depending on what you get, will decide next optimization step.
All the best
Chhattar Pal I suggest you to run a gradient. You are developing multiplex PCR and certainly you can do it in one go.
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