what are some general guidelines for manipulating Taq? - (Dec/21/2006 )
Could someone give me a general idea of the different sorts of manipulation one might use (perhaps refer me to a text or guide) to alter the results of a PCR reaction using Taq?
For example: how do I change [MgCl2], extension time, annealing temperature, hot start settings, concentration of other components (primers, dNTP's) etc. to achieve different objectives, such as:
1. higher specificity (particularly interested in this one)
2. higher sensitivity
3. higher yield of desired product
I'm sure this info is available somewhere in general form, and I'm aware that it will probably vary by primers/template.
primer concentration should be increased in case of long pcr as the primer is more sensitive than the template.
To decrease unspecific, you may play with TM (increase) or add DMSO.
If your primers hold secondary structures possibilities and dimerization capacities adding DMSO without changing the tm decrease possibilities of annealing
Thanks for your help,
I came across this, I think it contains some good information.