PCR: Polymerase Chain Reaction: Technique for copying DNA.
PCR uses: Template DNA (your original samples)
DNA polymerase:- Enzyme that copies DNA
-From Thermus Aquaticus (bacteria that live in hot springs;100 degrees Celsius)
- This enzyme does not have denatured proteins at this temperature.
-DNA primer is a swatch of DNA complementary to the gene you want to amplify.
-Other phosphorlyated bases (GTP, ATP, CTP, TTP)
How PCR works:
1.) Double stranded DNA strands seperate at 90 degrees Celsius
2.) When cool in presence of DNA polymerase and nucleotide triphosphates, DNA polymerase synthesizes complementary strand for each template strand that there's a primer for.
Steps:
1.) Get 5 PCR tubes
-Positive Control (plasmid that has GAPDH gene in it)
-Negative Control (sterile water; Should have NO DNA in it)
-Purified DNA from Arabidopsis Thaliana (plant known to have the gene)
-DNA from plant 1
-DNA from plant 2
2.) Add 20 microliters of MM (master mix: nucleotide triphosphates and DNA polymerase) to each tube.
3.) Add 15 microliters of sterile water to each tube
4.) Add 5 microliters of DNA to each tube (Positive, Negative, Purified, Plant 1 and Plant 2). Be sure to change pipette tips!
5.) The tubes were then left over night in the PCR machine.
