I'm doing PCR using primers with different melting temperature (60 and 66).? - primer annealing and melting calculation
What is the best annealing temperature, which gives the best result? How can problems with the melting of the different primer primer design again? Thank you people.
Wednesday, February 17, 2010
Primer Annealing And Melting Calculation I'm Doing PCR Using Primers With Different Melting Temperature (60 And 66).?
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4 comments:
PCR does not know what use the machine you are, but some possible temperature ramp (starting with their calculated lower annealing temperature and increase slightly with each cycle - namely, the increasing hardness). A gradient block is the best - so that you can try different combinations of both.
Empiracal take some tests. Continue to experiment and do not give you up!
A general rule-of-thumb is to use a hybridization temperature of 5 ° C below the melting temperature. Do you agree with the multiplex PCR with primers for these two men? If not, then it should not matter if the annealing temperature are different, which can be multiplied in PCR reactions. If you do), multiplexed (or with a PCR plate / thermocycler for all reactions, then I suggest that 55C and optimization.
55 would work.
This difference is not so important, but it is the melting temperature of the primer by the addition or deletion of nucleotides adapted nearer.
You can use the number that occurred in the gel.
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