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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
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Buy this book, M8265
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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 |
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| Chloroform |
C2432 |
Chloroform |
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| dNTP solution |
D7295 |
dNTP Mix, Solution of all four dNTPs |
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| MgCl2 |
M8787 |
Magnesium chloride solution PCR Reagent |
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| Placental RNase inhibitor |
R2520 |
Ribonuclease Inhibitor |
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| Sense and antisense primers |
Sigma-Genosys |
Sigma-Genosys |
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Polymerases
| Product Code |
Product Name |
Add to Cart |
| cDNA generation |
| A4464 |
Enhanced Avian (eAMV) Reverse Transcriptase |
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| STR-1 |
Enhanced Avian First Strand Synthesis Kit |
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| R9376 |
AMV Reverse Transcriptase |
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| M1302 |
M-MLV Reverse Transcriptase |
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| R7647 |
Random Nonamers (univ. lib. primers) |
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| SYBR based detection |
| S4438 |
SYBR® Green JumpStart™ Taq ReadyMix™ for QPCR |
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| S1816 |
SYBR® Green JumpStart™ Taq ReadyMix™ for QPCR, Capillary formulation |
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| S5193 |
SYBR® Green JumpStart™ Taq ReadyMix™ without MgCl2 |
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| S9194 |
SYBR® Green JumpStart™ Taq ReadyMix™ for High Throughput QPCR |
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| Probe based detection |
| D7440 |
JumpStart™ Taq ReadyMix™ for QPCR |
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| D6442 |
JumpStart™ Taq ReadyMix™ for for High Throughput QPCR |
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| Other QPCR reagents |
| R4526 |
Reference Dye for Quantitative PCR |
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| Integrated cDNA/DNA amplification |
| QR0100 |
SYBR® Green Quantitative RT–PCR |
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| 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.
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