PCR Optimization Flashcards

Flashcards for PCR optimization lecture review.

PCR Components

200 mM Tris pH 8.4, 500 mM KCl, 10 mM dATP, 10 mM dTTP, 10 mM dCTP, 10 mM dGTP, DNA polymerase, template DNA, forward primer, reverse primer, MgCl2

Tris pH 8.4 and KCl Role in PCR

Maintain proper enzyme folding and enzymatic activity by meeting optimal pH and salt requirements.

MgCl2 Role in PCR

Cofactor for DNA polymerase, stabilizes primer/template binding.

dNTPs Role in PCR

Building blocks for making new DNA molecules.

Template DNA Role in PCR

Contains the DNA sequence to be amplified.

Forward and Reverse Primers Role in PCR

Primers that DNA polymerase uses to extend in the 5'-3' direction on each strand of DNA.

DNA Polymerase Role in PCR

The macromolecular machine that performs the work of DNA synthesis.

Impact of High dNTPs in PCR

Binds to magnesium ions, effectively decreasing magnesium and destabilizing primer/template interactions.

Primer:Template Ratio Calculation

With 0.5 μM primer and 1x10^7 template molecules, there are 6 x 10^5 primer molecules for every template molecule.

PCR Product Calculation After 40 Cycles

Approximately 1x10^12 products from every template.

Impact of Increased Primer Concentration

Increases primer/primer interactions, leading to primer dimers.

Impact of Increased Template DNA Copy Number

Increases PCR products until primers are depleted or polymerase denatures.

Impact of Increased Primer/Template on Mg2+ ions

Will sequester Mg2+ ions and destabilize primer/primer and primer/template interactions.

Mass of 10^7 Plasmid DNA Molecules (5 kb)

Approximately 50 pg.

Taq DNA Polymerase Reaction

Catalyzes the addition of dNTPs to a DNA strand. Reaction: DNA polymer + dNTP → DNA polymer(+1) + PPi

Hot Start PCR

A technique that improves PCR specificity by inactivating the DNA polymerase until a high temperature is reached.