Power SYBR Green PCR Master Mix and RT-PCR Protocol (PN ...
Power SYBR Green PCR Master Mix and RT-PCR Protocol (PN ...
Power SYBR Green PCR Master Mix and RT-PCR Protocol (PN ...
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Chapter 4<br />
Optimizing Primer Concentrations<br />
Thermal Cycling<br />
Parameters for<br />
Primer<br />
Optimization<br />
Step<br />
AmpliTaq Gold ®<br />
Polymerase<br />
Activation<br />
<strong>PCR</strong><br />
Cycle (40 cycles)<br />
Denature Anneal/Extend<br />
HOLD<br />
CYCLE<br />
Temperature/<br />
Time<br />
95 °C<br />
10 min<br />
95 °C<br />
15 sec<br />
60 °C<br />
1min<br />
Volume 50 µL<br />
IMPO<strong>RT</strong>ANT! The 10 min, 95 °C step is required to activate the<br />
AmpliTaq Gold ® DNA Polymerase, LD.<br />
Confirm the<br />
Absence of<br />
Nonspecific<br />
Amplification<br />
To confirm the absence of nonspecific amplification:<br />
Step<br />
Action<br />
1. Analyze the <strong>PCR</strong> products by agarose gel electrophoresis.<br />
2. Generate a dissociation curve on the<br />
Applied Biosystems 7900HT Real-Time <strong>PCR</strong> System, the<br />
Applied Biosystems 7500 Real-Time <strong>PCR</strong> System, the<br />
Applied Biosystems 7300 Real-Time <strong>PCR</strong> System, the<br />
ABI PRISM ® 7700 SDS, the ABI PRISM ® 7000 SDS, or<br />
the GeneAmp ® 5700 SDS.<br />
4-6 <strong>Power</strong> <strong>SYBR</strong><br />
DRAFT<br />
® <strong>Green</strong> <strong>PCR</strong> <strong>Master</strong> <strong>Mix</strong> <strong>Protocol</strong><br />
September 29, 2005 1:24 pm, 4_Chapter.fm
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