Parameters involved in the conversion of real-time PCR assays from the ABI prism 7700 to the Cepheid SmartCycler II.
OBJECTIVE
We examined several critical parameters that must be optimized when converting between the ABI Prism 7700 real-time PCR platform and the Cepheid SmartCycler II while using the same primer and probe sequences.


METHODS
A lyophilized master mix, MgCl(2) concentration, PCR cycling conditions, and ramp times were evaluated.


RESULTS
Optimization of each parameter, including use of the OmniMix HS-lyophilized beads, 6 mM MgCl(2) concentration, changes in PCR cycling parameters, and increased ramp time were necessary to convert this real time PCR assay to a new platform.


CONCLUSIONS
We conclude that careful consideration of several analytical parameters can result in a smooth transition of assays between real time PCR platforms.
Bentley
Heather A
HA
Department of Pathology, Dartmouth Medical School, Dartmouth-Hitchcock Medical Center, One Medical Center Drive, Lebanon, NH 03756, USA.
Belloni
Dorothy R
DR
Tsongalis
Gregory J
GJ
D007202:Indicators and Reagents; D015636:Magnesium Chloride; D016133:Polymerase Chain Reaction / Q000295:instrumentation* / Q000379:methods / Q000592:standards*; D013696:Temperature; D013995:Time
D007202:Indicators and Reagents; D015636:Magnesium Chloride