Lab 7 Protocol Quiz (PCR & isolating genomic DNA)

The goal of the lab

• Confirm mutation of interest was generated in yeast that was transformed in lab 6.

• Genomic DNA will be isolated from transformed yeast cells and the MSH2 gene will be amplified using PCR reaction.

The critical components of PCR

DNA template ; 2 primers ; nucleotides ; DNA polymerase

First step of PCR

Denature. Heat reaction to 94°C-98°C. Allows DNA to separate into two single strands of DNA

Second step of PCR

Anneal. Cool reaction mix 48-65°C allows DNA primers complimentary to region of target DNA to anneal on each side of target region.

Third step of PCR

Extension. Heat sample up to 72°C ; provides optimal temperature for Taq DNA polymerase activity.

What is Taq Polymerase?

DNA polymerase that doesn’t denature in extreme heat.

Why Taq polymerase is used in PCR

Since it is heat resistant, it makes it so we wouldn’t have to add a new DNA polymerase every time the reaction was heated to 94°C in the first step of PCR.

Why Isopropyl Alcohol is added (step 10 in the protocol). 

In order to to precipitate (dissolve) the genomic DNA so it forms the pellet

What does PCR stand for?

Polymerase chain reaction