Polymerase Chain Reaction PCR

It is a fast and inexpensive technique used to AMPLIFY small fragment of DNA to Produce million of copies.

What is PCR used for?What is PCR used for? Diagnosing acquired or inherited diseases. Diagnosing viral or bacterial diseases.

Two Primers

DNA polymerase

Deoxy-nucleotide triphosphate (dNTP)

Buffer solution

MgCl2

Extracted DNA sample

Denaturation Step:

Heating the reaction to 90OC-95OC.

Annealing Step:

Rx temperature lowered to 50OC-56OC.

Extention/Elongation: 70OC-72OC.

30-40 cycles/ Automated using Thermal

Cycler.

taq

Objective:To make millions of copies of a specific DNA

segment consisting of MTHFR gene.

Materials Given:3 samples:1.Normal2.Homozygote3.Heterozygote

Volume for 1 Rx Volume for 5 Rxs

PCR Buffer 5μl 5x5=25μl

dNTPs 1μl 1x5=5μl

Forward Primer 1μl 1x5=5μl

Reverse Primer 1μl 1x5=5μl

Taq polymerase 0.2μl 0.2x5=1μl

dd.H2O 39.8μl 39.8x5=199μl

TOTAL VOLUME 48μl 240μl

Master MIX

DNA 2μl

TOTAL VOLUME 50μl

++

94oC

55oC

72oC

4oC

30 sec

30 sec

30 sec

∞

33 cycles