Lab Seven: Polymerase Chain Reaction

PCR! Who done it??

As a kid, I loved mystery books! Loved them! So when this lab came around, I was excited!

Mystery in the Molecular Cell Lab!

I think it was Dr. Cruz... in the lab... with the graduated cylinder!

What's important in this lab?

• What is PCR?
• What is Taq Polymerase?
• What are the components of PCR?
• What are the Steps of PCR? Where do they take place?
• Primers are...
• What is a genetic repeat? Polymorphism? Short Tandem Repeats?
• Why do we want so many copies?

PCR

-An alternative to cloning. It amplifies specific gene sequences by using Taq Polymerase and sequence specific primers.

Taq Polymerase was isolated from Thermus aquaticus and is thermostable (not denatured by heat)! So why do we use Taq?

PCR Components include:

• DNA (genomic or cDNA)
• Taq Polymerase
• SEQUENCE SPECIFIC Forward and Reverse Primers
• Deoxynucleotide Triphosphates (dNTPs)
• Reaction Buffer

STEPS!!!

-Denaturation: 95 degrees! HOT HOT HOT!

-peels double stranded DNA into single stranded DNA for copying!

-Annealing: 55-65 degrees!

-SEQUENCE SPECIFIC forward and reverse primers bind to ssDNA on sequence before target sequence

-Elongation: 72 degrees!

-Taq binds to primer and elongates the strand

REPEAT 20-30 times!

Make BILLIONS of copies!

Check out this youtube video! Super Boring, but does the trick!

Know what a Polymorphism is! A Short Tandem Repeat!

Don't get it? Post Questions!

What do our gels tell us?

Compare the Crime Scene DNA bands to Suspects 1-4 bands. Any near matches?

Any this is just for fun....I suggest watching it!

Biologists have too much free time while waiting for the thermocycler....