PCR/Amplification

Polymerase Chain Reaction (PCR) Basics

 What is PCR?

The polymerase chain reaction (PCR) is a powerful core molecular biology technique. It is an efficient and rapid in vitro method for enzymatic amplification of specific DNA or RNA sequences from various sources. PCR was developed by Kary Mullis and his colleagues in 1983 and they received the Nobel Prize in Chemistry in 1993 for their work.


 How does PCR work? What are the steps of PCR?

A simple PCR reaction consists of target DNA, a set of synthetic oligonucleotide primers that flank the target DNA sequence, a thermostable DNA polymerase (usually Taq polymerase), and nucleotides. There are three stages to each amplification cycle. The first step is denaturation, during which the DNA is heated to 90-95˚C double-stranded DNA (dsDNA) separates to single-stranded DNA. The temperature at which 50% of the dsDNA is denatured is known as the melting temperature (Tm) and is determined by the G+C content, the length of the sample, and the concentration of ions (primarily Mg2+). During the second step, annealing, the sample is cooled to 40-60˚C, which allows the primers to attach to the target DNA. The final step occurs at 70-75˚C and is known as extension. During this phase, DNA polymerase extends the DNA from the primers, creating new dsDNA with one old strand and one new strand. Because the strands synthesized in one cycle serve as a template in the next, a million-fold increase in the amount of DNA is achieved in just 20 cycles. These steps can be performed automatically using machines called thermal cyclers.1,2,3
 

 Enhanced PCR


Popular Products

REDTaq® ReadyMix™  |  Taq Pol + Mg Kit  |  Taq Pol Kit  |  REDTaq Kit


Routine PCR amplification is used to produce an amplified amount of DNA for down-stream applications including clone length verification. Our products offer a variety of tools to perform routine amplification. Particularly for convenience, we offer ReadyMix and REDTaq options.   

Taq DNA Polymerases

Taq DNA polymerase is a specialized thermostable enzyme isolated from Thermus aquaticus, a thermophillic bacterium. It is the most commonly used DNA polymerase for PCR and now the recombinant form is expressed in E. coli. The enzyme is capable of 5’ -> 3’ polymerase and exonuclease activity; SDS-PAGE examination finds no detectable contamination associated with endonucleases or exonucleases.
 

Product No.
Product Name Features
D1806 Taq DNA Polymerase with 10X Reaction Buffer • Buffer optimized with MgCl2
D4545 Taq DNA Polymerase with 10X Reaction Buffer without MgCl2 • Separate vial of MgCl2 provided

 

 REDTaq DNA Polymerases

REDTaq is exclusive to Sigma-Aldrich. It is a unique blend of Taq DNA polymerase with inert red dye. This blend performs as well as a clear Taq enzyme blend and does not interfere with downstream applications. It’s primary function is identification – solutions are easy to keep track of in PCR tubes and gels.  If necessary, the dye can be removed by standard purification methods post amplification.

REDTaq yield compared to standard Taq under identical conditions

Forget about the loading buffers and tracking dyes

  • REDTaq dye migrates like a 125 bp fragment
Product No. Product Name Features
D4309 REDTaq DNA Polymerase • Buffer optimized with MgCl2
D8312 REDTaq Genomic DNA Polymerase • For Genomic DNA amplification
• Includes 10X buffer optimized with MgCl2
D2812 REDTaq Genomic DNA Polymerase without MgCl2 • For Genomic DNA amplification
• Includes 10X buffer and separate vial of MgCl2
D6063 REDTaq SuperPak™ DNA Polymerase • High visibility polymerase for Genomic DNA
• Includes dNTPs and 10X MgCl2 optimized PCR buffer

 

 

ReadyMix


ReadyMixes are Taq DNA polymerase mixes that contain Sigma-Aldrich’s high quality Taq DNA polymerase, 99% pure dNTPs, and buffer, all in a 2X optimized reaction concentrate. This product is made for convenience – reduce pipette steps and minimize risk of contamination by eliminating various mixing steps. Simply add template and primers to the ReadyMix. ReadyMixes are formulated to address various PCR needs and can be purchased in combination with REDTaq for further convenience.


Amplification of 1, 2, 3, and 7 kb fragments and a
4.5 kb human genomic DNA using ReadyMix Taq
PCR Reaction Mix

 

Product No. Product Name Features
R2523 REDTaq ReadyMix • Includes PCR Grade Water reagent
• Includes REDTaq dye for identification
P4600 ReadyMix Taq PCR • Includes PCR Grade Water reagent

References

  1. Innis, Michael A., et al., eds. PCR protocols: a guide to methods and applications. Academic press, 2012.
  2. Haynie, Donald T. Biological thermodynamics. Cambridge University Press, 2001.
  3. McPherson, Mike, and Simon Møller. Pcr. Taylor & Francis, 2000.