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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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Real-time PCR Components

To perform Real-time PCR, the extracted nucleic acid (NA) is combined with individual reagent components necessary to drive the reaction. These components consist of the NA extract along with primers, dNTPs (purine and prymidine bases), and a polymerase enzyme. The mixture is optimized with buffer and additional reagents to stabilize and drive the reaction.
Discription of reagents:
  • Primers – Short fragments of oligodeoxynucleotides, 18-25 bps in length, that flank the target sequence.
  • Thermostable Polymerase – Responsible for extension.
  • Enzyme Cofactors – Works with the polymerase, generally magnesium.
  • Free dNTPs – The actual building blocks for DNA.
  • Stabilizer – Includes buffer, glycerol, KOH, etc. stabilizes the reaction.