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 2<br />
<strong>PCR</strong><br />
Amplifying Custom Target Sequences for<br />
Quantitation<br />
Overview<br />
We recommend the following steps for the development of real-time<br />
quantitative <strong>PCR</strong> assays:<br />
Step Action See Page<br />
1. Order Reagents 2-4<br />
2. Quantitate Primers 2-4<br />
3. Optimize Primer Concentrations for:<br />
• <strong>PCR</strong><br />
• One-Step <strong>RT</strong>-<strong>PCR</strong><br />
• Two-Step <strong>RT</strong>-<strong>PCR</strong><br />
4-3<br />
4-9<br />
4-13<br />
Ordering<br />
Reagents<br />
Quantitating<br />
Primers<br />
See “Materials Required but Not Supplied” on page 1-6 for a list of<br />
required reagents <strong>and</strong> equipment.<br />
Use a spectrophotometric method to determine the concentrations of<br />
the primers received:<br />
• Measure the absorbance at 260 nm of a 1:100 dilution of each<br />
oligonucleotide in TE buffer.<br />
• Calculate the oligonucleotide concentration (C) in µM using the<br />
method shown in the table on the next page.<br />
2-4 <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, 2_Chapter.fm
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