Home made quantitative real time RT-PCR? SYBR Green?

Dr. Daktari dr.daktari at gmx.net
Tue Jul 2 07:40:55 EST 2002


We have a MJResearch Opticon I and are very happy with it. We have set
up an in house SYBR Green 1 assay for several different RNAs using in
principle standard reaction conditions. The Mg concentration was
increased with 1 mM due to the presence of SYBR green. Our final
amount of SYBR green is 0.5x from the 10,000 stock from Sigma or from
Biowhittaker Molecular Applications.

SYBR green is much cheaper but gives in general big problems with
primer dimers and small products. Slightly larger products with higher
Tm's are recommended.

idelgado at bcm.tmc.edu wrote in message news:<20020627221947.11893.qmail at ww02.hostica.com>...
> Dear all,
> 
> We just obtained a new ABI 7000 quantitative real-time RT-PCR machine and are in the process of deciding what kind of chemistry to use for our assays. We would like to try Sybr Green first and see if we can get the data we need without having to use Taqman probes.
> 
> My question is: does anybody out there have experience with the amount of Sybr green that needs to be used per reaction?. We would like to set up our own reactions and the sole component that we have no idea about how to use is Sybr Green. Any help in this regard is greatly appreciated.
> http://www.biowww.net/index.php/forum/forumlist/1/



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