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Annealing Temperatures for PCR
I totally agree with Jai - you really need to run your sequences thru a
calculator to get a GENERAL idea and then run PCRs with a range of
annealing tempuratures (I LOVE my gradient thermocycler). Then you will
want to optimize the amount of Mg++ and the amount of DNA added for your
specific type of samples.
* plant DNA seems to prefer ~4mM MgCl, while the bacteria I work with
amplifies best with 1.5mM.
* some DNA extraction protocols will leave left-over substances that are
inhibitory to PCR thus less of the DNA prep must be used to get good
amplification (this is really counter-intuitive when you see light bands
being produced). Too much DNA will throw off the PCR reaction also.
Good luck, hope this helps
|annealing , pcr , temperatures|
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