Genomic DNA isolation and PCR

 What is the purpose of binding buffer, wash buffer, elution buffer, the silica column, RNase A, and proteinase K?

Binding buffer: makes DNA stick to silica.

Wash buffer: removes salts/proteins.

Elution buffer: releases DNA from silica.

Silica column: binds nucleic acids under high salt.

RNase A: removes RNA.

Proteinase K: digests proteins.

 What are the three reagents, quantities, and purposes used to make 50 mL of a 1% agarose gel? What allows you to visualize DNA?

1% gel →0.5 g agarose

50 mL TAE/TBE buffer

Ethidium Bromide allows us to see the stained DNA

 What technique would you use to visualize whole genomic DNA?

Visualizing DNA

Use agarose gel electrophoresis with a DNA stain + UV light.

 What is the purpose of adding loading dye to your isolated DNA sample?

Loading dye purpose, Adds weight, color, and tracking so DNA sinks & migration is visible.

 What does PCR stand for?

stands for polymerase chain reaction

 What is the purpose of PCR?

amplify specific DNA region

 What are some applications of PCR (why would you use it)?

applications: diagnostics, cloning, sequencing, forensics, etc.

 What are the three basic steps of PCR and what occurs during each?

Denaturation(heated to break H bonds on complementary strands),

Annealing (cooling, to allow the primer to bind to the complementary sequence, and

Elongation (heating to allow the polymerase to bind and synthesize new nucleotides off of the nucleotides.

 What are the purposes of the four major components of the PCR reaction? What needs to change when looking at two different genes?

H2O,

2x Go-Taq master mix,

F primers

R primers

The primers need to be different for each gene.

 What determines the section of DNA that is amplified?

Primers

 Why is it important to not include your DNA sample into your master mix?

It will react befor it does with the sample DNA, and contaminate.

 When you construct your master mix, how do you determine the number of reactions you should calculate for?

2^N where N is the number of cycles of PCR performed.

 What are some errors that could occur when you run a PCR gel electrophoresis?

How would those errors look on the gel image? No band, to many, or smear

 Know how to design a forward and reverse primer when given a sequence of interest.

Forward is 5 to 3 prime RNA bound to non gene side, reverse is opposite.