Molecular Biology

Quantitative PCR Protocol

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Selected Protocol from "Molecular Cloning: A Laboratory Manual", Third Edition, Joseph Sambrook, David W. Russell.

Quantitative PCR

Joseph Sambrook
Peter Maccallum Cancer Institute and The University of Melbourne, Australia
David W. Russell
University of Texas Southwestern Medical Center, Dallas

Buy this book, M8265

ABSTRACT
Quantitative PCR involves co-amplification of two templates: a constant amount of a preparation containing the desired target sequence and varying amounts of a reference template. After amplification, the concentration of the target sequence in the preparation of nucleic acid under test is established by interpolation into a standard curve. Quantitation of nucleic acids by PCR is best performed by real-time PCR (see MC3, pp. 8.94–8.95). However, the following robust protocol, which uses radioactivity to quantify PCRproducts, remains useful when a real-time instrument is unavailable. The method can be easily adapted to other methods of quantification such as fluorometry.

Products Available for this Protocol

Protocol Material Description Product #  Product Name Add to Cart
Reagents and Solutions      
Chloroform C2432 Chloroform
dNTP solution D7295 dNTP Mix, Solution of all four dNTPs
MgCl2 M8787 Magnesium chloride solution PCR Reagent
Placental RNase inhibitor R2520 Ribonuclease Inhibitor
Sense and antisense primers Sigma-Genosys Sigma-Genosys  

Polymerases

Product Code Product Name Add to Cart
cDNA generation
A4464 Enhanced Avian (eAMV) Reverse Transcriptase
STR-1 Enhanced Avian First Strand Synthesis Kit
R9376 AMV Reverse Transcriptase
M1302 M-MLV Reverse Transcriptase
R7647 Random Nonamers (univ. lib. primers)
SYBR based detection
S4438 SYBR® Green JumpStart™ Taq ReadyMix™ for QPCR
S1816 SYBR® Green JumpStart™ Taq ReadyMix™ for QPCR, Capillary formulation
S5193 SYBR® Green JumpStart™ Taq ReadyMix™ without MgCl2
S9194 SYBR® Green JumpStart™ Taq ReadyMix™ for High Throughput QPCR
Probe based detection
D7440 JumpStart™ Taq ReadyMix™ for QPCR
D6442 JumpStart™ Taq ReadyMix™ for for High Throughput QPCR
Other QPCR reagents
R4526 Reference Dye for Quantitative PCR
Integrated cDNA/DNA amplification
QR0100 SYBR® Green Quantitative RT–PCR
QR0200 Quantitative RT–PCR ReadyMix™ for probe based applications

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Product Association Disclaimer: The Sigma-Aldrich products listed for this specific protocol were selected either to match or to supplement the products listed within the actual protocol. The products/reagents from Sigma-Aldrich have been qualified for usage, but may not have been validated for this specific application. Please refer to the detailed product description on the usage of specific products of interest.