Taqman Assay Question

The Lab Rat thelabrat at thelabrat.com
Wed Mar 1 15:56:29 EST 2000


Dave Knorr wrote in message ...
>
>PE also has a pretty good piece on their website about optimization.
>Briefly, their philosophy is to give the enzyme everything it needs to be a
>good polymerase, and then change the primer sequence and concentrations to
>optimize the conditions. The most common approach to PCR optimization seems
>to be optimizing the reagents and cycling conditions to the primer.  By
>holding the reagents and cycling constant, any assay can be run with the
>same mix, and assays can be run side-by-side.  The optimization for any
>TaqMan system take only a couple of runs.  True, you might find the overall
>performance of that assay to be less than you'd hoped for, but it will be
>optimized.  You'll have to develop another primer/probe set that you hope
>will be better.
>
>Dave Knorr
>

Dave


In practice, a lot of times you are constricted to a small amplicon.  This
is especially true with respect to the original question of allele
discrimination!  I would agree that the optimization approach PE takes is
great if you have several kb to work with and could design several
primer/probe sets, but it is not acceptable when you have 200bp or less to
design your assays and you don't have the luxury to follow "the rules".

Basically, this is the whole "reasoning" behind their Universal master mix.

I do not usually deviate from the 60C recommended for Taqman (unless doing
allele discrimination), but 30% of the assays I have developed do not
operate optimally using the UMM conditions (% is higher for RT-PCR
reactions) and we still have to a traditional Mg and primer/probe
concentration matrix.

What has been your experience???

JeffF

-----------------------------------------------------------------------
Jeff Fairman, Ph.D.
Senior Scientist, Pharmacogenomics Research
Clingenix, Inc.
871 Industrial Road, Suite J
San Carlos, CA 94070
(650) 598-7645 (office)
(650) 598-7641 (fax)
jfairman at clingenix.com


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