Preparation of PCR Reaction

The PCR master mix contains all of the components necessary to make new strands of DNA in the PCR process. The master mix reagents include:

Final Conc.

Component

Purpose

1X

Buffer

Maintains proper pH of the PCR reaction.

200 |M

Deoxynucleotides

Provide both the energy and nucleosides for the synthesis of DNA. It is important to add equal amounts of each nucleotide (dATP, dTTP, dCTP, dGTP) to the master mix to prevent mismatches of bases.

0.2-1.0 |M

Primers

Short pieces of DNA (20-30 bases) that bind to the DNA template allowing Taq DNA polymerase enzyme to initiate incorporation of the deoxynucleotides. Both specific and universal (arbitrary) primers can be used.

1.5-2.5 |U

Taq polymerase

A heat-stable enzyme that adds the deoxynucleotides to the DNA template.

<1.0 |g

Template DNA

The DNA amplified by the PCR reaction.

Master mix buffer is often stored as a 10X stock solution (100 mM Tris-HCl, pH 8.3,500 mM KCL, 1.5 mM MgCl2) and diluted to 1X for use. Both the master mix buffer and the purified water can be stored at room temperature. Oligonucleotides (primers and probes), enzyme, and dNTPS should be stored at -20°C or according to manufacturers', recommendations.

Depending on the PCR platform and the configuration of the reaction tubes, the PCR reaction volume can vary greatly. Regardless of the reaction volume, the concentration of the individual components should remain constant.

Master mix reagents can be obtained from a number of vendors. Initial concentrations vary, and it is important to read the specifications carefully and make appropriate dilutions. The formula used to determine volume of a stock reagent is as follows:

(initial concentration) x (volume needed) = (final concentration)

Master mix kits containing ready to use reagents are available from a number of vendors. These kits benefit the molecular diagnostic laboratory by providing standardization, decreased preparation time, and reduced risk of contamination.

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