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The page below is a sample from the LabCE course Real-Time PCR. Access the complete course and earn ASCLS P.A.C.E.-approved continuing education credits by subscribing online.

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Reaction Mixture

The first step in a PCR procedure is the creation of a reaction mixture. This mixture should consist of the DNA template that is to be amplified, or genomic DNA, along with two different single stranded primers, one for each flanking side of the DNA of interest. Also added to this mixture is the polymerase, an enzyme cofactor, deoxyribonucleotide triphosphates (dNTPs), and a stabilizer or buffer.

The following is the complete list of components needed for a PCR reaction:

  • Primers – Short fragments of oligodeoxynucleotides, 20-30 bps in length, that define the target sequence.
  • Thermostable Polymerase – Responsible for extension.
  • Enzyme Cofactor – Works with the polymerase, generally magnesium.
  • Free dNTPs – the actual building blocks for DNA
  • Stabilizer – Includes buffer, glycerol, KOH, etc. Stabilizes the reaction.