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Published on: May 22, 2012
Real-time PCR (qPCR) primer design using free online software
Brenda Thornton1, Chhandak Basu
1School of Biological Sciences, University of Northern Colorado, Greeley, Colorado 80639, USA.
Summary
This guide explains quantitative PCR (qPCR) and its fluorescent chemistries, focusing on SYBR® Green dye. It provides a protocol for designing effective SYBR® Green primers using free online tools for gene expression analysis.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- Quantitative PCR (qPCR) is the standard for validating gene expression profiles.
- qPCR utilizes reverse transcription PCR (RT-PCR) with fluorescent chemistry to quantify gene expression.
- Common chemistries include SYBR® Green dyes and TaqMan®, Molecular Beacon, or Scorpion probes.
Purpose of the Study:
- To detail the principles of real-time PCR fluorescent chemistries.
- To outline essential primer design rules and common pitfalls for SYBR® Green assays.
- To provide a practical protocol for designing SYBR® Green primers using free online software.
Main Methods:
- Explanation of SYBR® Green dye chemistry and its reliance on primer design.
- Discussion of online primer design software for SYBR® Green applications.
- Step-by-step laboratory protocol for primer design.
Main Results:
- SYBR® Green offers a cost-effective and simple method for qPCR.
- Successful SYBR® Green qPCR is highly dependent on optimal primer design.
- Free online tools facilitate the design of effective SYBR® Green primers.
Conclusions:
- Effective primer design is critical for reliable SYBR® Green qPCR results.
- Accessible online tools empower researchers to design SYBR® Green primers.
- This exercise serves as a foundational guide for qPCR primer design.
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