PCR preparation
To perform a PCR experiment you need:
- Primers
- Nucleotides
- DNA polymerase
- DNA template
- Extra care with contaminations
- A thermocycler
If any of these components is missing no reaction will occur and no DNA will be synthesized.
Master mixes contain most of the PCR components (except for the DNA template) in a larger quantity - relative to the amount of PCR reactions we would like to do. You can reduce the experimental error and variability and save time by preparing master mixes.
They usually contain the specific forward and reverse primers, the nucleotides (dNTPs), the Taq polymerase enzyme, and optionally MgCl2, nuclease-free water, and a reaction buffer.
Primers: primers will bind at a specific DNA sequence and mark the beginning of the DNA amplification. Primer design is very important to successfully amplify the region of interest. They define the length of the PCR product by limiting the sides of it.
Nucleotides: nucleotides are required to build the new DNA sequence, they are the DNA building blocks.
Polymerase: polymerase is the enzyme that assembles the nucleotides based on the template sequence. Taq polymerase is the most common DNA polymerase utilized in PCR experiments.
DNA template: a template is required to be the basis of new DNA sequence amplification. Perform DNA isolation to extract DNA from cells.
Extra care with contaminations: when preparing for a PCR experiment, you must be extra careful of potential contamination. PCR is a very powerful technique to amplify DNA. This means that if you have a tiny contamination (for example, DNA coming from other samples), this DNA can also be amplified, competing with the original template and destroying your experiment's results. To prevent contamination, you need to always use gloves and work in a a very clean environment (for instance, change the pipette tips when you are pipetting from a different container, tie your hair or do not cough or sneeze around the PCR workbench).