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-   -   Annealing Temperatures for PCR (http://www.molecularstation.com/forum/protocols-methods-forum/18375-annealing-temperatures-pcr.html)

Deanne Bell 03-30-2006 04:01 PM

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.


For example:
* 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
Deanne Bell








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