Taqman Assay Question

The Lab Rat thelabrat at thelabrat.com
Mon Feb 28 11:01:23 EST 2000


MAG wrote in message <38BA82C5.D445CB27 at mail.gov>...
>Hello,
>We have recently obtained a 7700 SDS machine from PE.
>We want to do allelic discrimination assays.
>My question is does anyone have a general protocol for the set up of the
>PCR master mix and thermal cycler conditions?
>Such as the amounts of primers, probes, buffer dNTPs, etc.
>We would like to stay away from the Universal Master mix they are
>pushing, but they are not offering any technical support for assays that
>do not use their Universal Master mix.
>This is sad, considering the amount we have already paid for the unit.
>
>
>Thank You
>
>
>Michael Gandolph
>NIH/NCI/DCEG/LPG
>41 Library Drive
>Building 41  Room D 702
>Bethesda, MD  20892
>E-Mail:  gandolpm at mail.nih.gov
>Telephone:  (301) 402-5846
>
>

Michael,

Check this reference for a general 'homebrew' master mix.  We have also used
this master mix for allelic descrimination as well as RT-PCR.

BioTechniques 27:  566-574 (September 1999).

You have to tweak the allele discrimination assays a little more than
normal.  I would suggest the standard Mg vs. primer concentration matrix,
followed by +/- 2C on the extension step (start at 60C).  The easiest way is
to design as short an amplicon as possible and to order "standard" oligos
which conform to your alleles and you can then PCR and clone these.  Then
you can use this product for your assay development.


JeffF

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