Quick Guide: Polymerase Chain Reaction (PCR)
What is the Polymerase Chain Reaction (PCR)?PCR is a technique that allows researchers to quickly create many copies of a specific region of DNA in vitro.
What Do I Need to Perform PCR?
TEMPLATE - the purified, double-stranded piece of DNA we want to copy
PRIMERS - short synthetic DNA molecules that target a specific DNA sequence for amplification
TAQ DNA POLYMERASE - thermostable enzyme used to copy DNA
FREE NUCLEOTIDES - the building blocks of DNA
THERMAL CYCLER (a.k.a. PCR machine) - a specialized machine that rapidly heats and cools the samples
How Does PCR Work?
To perform PCR, the template is mixed with primers, Taq polymerase and nucleotides. The mixture is heated to 94°C to denature the DNA duplex (i.e., unzip it into single strands). Next, the sample is then cooled to 45°C-60°C, allowing the primers to base pair with the target DNA sequence (called “annealing”). Lastly, the temperature is raised to 72°C, the optimal temperature at which Taq polymerase will extend the primer to synthesize a new strand of DNA. Each “PCR cycle” (denaturation, annealing, extension) doubles the amount of the target sequence in less than five minutes. In order to produce enough DNA for analysis, twenty to forty cycles may be required.
- 1. ADD 20µl specific primer mix, 5µl extracted DNA and the EDVO PCR bead to a labeled 0.2 mL tube.
- 2. MIX the PCR sample. Make sure the EDVO PCR bead is completely dissolved.
- 3. CENTRIFUGE to collect the sample at the bottom of the tube.
- 4. AMPLIFY DNA using PCR:
- 5. PLACE tubes on ice. Analyze samples using agarose gel electrophoresis.
PROCEED to Performing Electrophoresis