Cell Bio Labs 9 and 10 - DNA Isolation and PCR - Review

Polymerase chain reaction (PCR)

In this lab, we amplified the GAPDH gene of the genomic DNA using this process. This is used to amplify a short segment of DNA with primers specific to the host

Breast cancer cells (MDA-MB-231) which are late stage/metastatic

Which cells were used in this lab?

GAPDH

Also known as glyceraldehyde-3-phosphate dehydrogenase, this catalyzes the 6th step in glycolysis and is involved in breaking down sugars for energy. The expression of this is routinely used in molecular biology labs as a reference/housekeeping gene to analyze the expression of all other genes

Housekeeping genes

These are defined as constitutive genes that are required fro the maintenance of cell function, and are not altered in most physiological or pathophysiological conditions

10ng/μL

What should the DNA sample be diluted to for the final PCR concentration?

There needs to be the same amounts of DNA loaded into the PCR, so the concentrations of all samples need to be the same

Why is it important that the DNA be diluted to a specific concentration for PCR?

Nucleotides (A, T, C, G), DNA polymerase, MgCl2, water

What makes up the Mastermix used?

Master mix, GAPDH (forward primer), GAPDH (reverse primer), DNA, and nuclease free water

What components make up the solution that goes through the PCR?

1) Denaturation (94°C)

2) Annealing (60°C and primers attach around 70°C)

3) Elongation (72°C)

What are the three parts of PCR and what temperatures do they occur at?

It is required for polymerase function in an isolated system

What is the function of MgCl2 in Mastermix?

M1V1=M2V2

V2 = 2.85 μL

If the initial concentration of DNA is 28.1 ng/μL, the final concentration should be 10 ng/μL, and the final volume is 8 μL, what is the initial volume that should be used?

They are needed to initiate and allow DNA replication of the target gene. They are specific for the host

Why are primers important?

MgCl2 because it is required for polymerase function in the isolated system (Check if correct)

What is present in the GoTaq Green Master Mix that is used to produce amplification during the PCR?

Equal concentrations allow the 5' to 3' strand to be replicated in the same amount as the 3' to 5' strand. The single strands produced from either template need to be present in the same amounts. (check if correct)

Why do the forward and reverse primers have the same final concentrations in the PCR mix?

It is a DNA polymerase that works at a higher temperature and adds nucleotides after the primers

What does the Taq polymerase do?

One forward primer and one reverse primer

The two primers used in the PCR reaction are. . .

Only one target sequence is acquired at the end of PCR, but it is replicated numerous times (check if correct)

How many target sequences are acquired at the end of a polymerase chain reaction?

Mastermix

What solution are the free nucleotides and Taq polymerase found in?