Polymerase Chain Reaction

In vivo

Replication takes place in a cell, it's a highly regulated and complex, aiming for complete abd accurate genome duplication

In vitro

Replication takes place in a test tube, PCR is a simplified and amplified version of targeting a specific DNA sequence for rapid replication. This pace of replication allows us to study DNA replication more efficiently in molecular biology.

Denaturation

Denatures double-stranded DNA (Breaks hydrogen bonds between base pairs)

Heated at 94 degrees Celsius

Annealing

Allow primers to anneal to template strand

Cooled at 55 degrees Celsius

Extension

Template strand extend from 3' end of primers

Mixed to 72 degrees Celsius (optimal temperature for Taq Polymerase)

Sterile water

Diluent; allows for adjustment of the final volume and concentration of the other components in the master mix

10X PCR Buffer

Provides optimal environment and pH for the Taq polymerase enzyme to function most effectively

dNTPs

Provide the individual nucleotides that Taq polymerase uses to synthesize new DNA strands complementary to the template DNA

Primers

Short, single-stranded DNA sequences that act as starting points for DNA synthesis. Taq polymerase enzyme can only add nucleotides to an existing 3' OH- group, which primers provide

Taq DNA polymerase

Thermostable enzyme that synthesizes new DNA strands complementary to the template DNA in PCR.

pGLO plasmid

serves as the template DNA, contains a gene that confers ampicillin resistance