ABI Prism® 7900HT Sequence Detection System ... - OpenWetWare

Assay Development Guidelines
TaqMan Assay
Development
Program
Identify Target
Sequence(s)
C-2 Designing TaqMan Assays
To develop custom TaqMan 5´ nuclease assays:
Step Action See Page
1 Identify target sequence(s). C-2
2 Design the TaqMan ® probes and the forward and reverse primers. C-3
3 Order reagents. C-4
4 Quantitate the concentrations of the probes and primers. C-4
5 Prepare the master mix. C-4
6 Optimize the primer concentrations. C-4
7 Run the assay. C-4
A target template is a DNA, cDNA, RNA, or plasmid containing the nucleotide
sequence of interest. For optimal results, the target template should meet the
following requirements:
♦ The target nucleotide sequence must contain binding sites for both primers
(forward and reverse) and the fluorogenic probe.
♦ Short amplicons work best. Amplicons ranging from 50–150 bp typically yield the
most consistent results.
♦ If designing assays for quantitative PCR, see “Design Tips for Quantitative PCR
Assays” on page C-6 for additional recommendations.