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 ...
You also want an ePaper? Increase the reach of your titles
YUMPU automatically turns print PDFs into web optimized ePapers that Google loves.
Chapter 4<br />
Optimizing Primer Concentrations<br />
Optimizing Primer Concentrations for <strong>PCR</strong><br />
Overview<br />
The purpose of the procedure below is to determine the minimum<br />
primer concentrations giving the lowest threshold cycle (C T ) <strong>and</strong><br />
maximum ∆R n while minimizing nonspecific amplification. The<br />
reaction volumes are 50 µL. Use 10 to 100 ng of genomic DNA or 1<br />
to 10 ng of cDNA template.<br />
Use <strong>PCR</strong> <strong>Master</strong> <strong>Mix</strong> to run four replicates of each of the nine<br />
conditions shown in the table below. The master mix is described in<br />
“<strong>PCR</strong> <strong>Master</strong> <strong>Mix</strong> for Primer Optimization” on page 4-4.<br />
Reverse Primer<br />
(nM)<br />
Forward Primer (nM)<br />
50 300 900<br />
50 50/50 300/50 900/50<br />
300 50/300 300/300 900/300<br />
900 50/900 300/900 900/900<br />
4-2 <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
Change language