Lecture 4 Pt. 1

Question 1

How can you separate DNA fragments based on mass?

Answer 1

DNA gels (add in ethidium bromide to be a fluorescent molecule)

Question 2

What can endonucleases do?

Answer 2

Restrict DNA, they recognize certain sequences and all for water to cleave the phosphate backbone

Question 3

How can we sequence DNA?

Answer 3

ddNTPs

Question 4

What are the steps to polymerase chain reaction?

Answer 4

1. separate strands by heating, cooling, and anneal primers
2. Extended by DNA polymerase with dNTPs
3. The new strands join together, then separate these strands by heating, cool, and anneal more rpimers
4. Extend primers by DNA primers again
5. Do this cycle over and and over

Question 5

Describe the process of mutating DNA

Answer 5

1. Make a weird primer with desired changed bases to be lifted
2. Add to the gene you want to be altered
3. DNA polymerase extends the mismatched primer to generate a mutated gene

Question 6

What do SDS PAGE Gels do?

Answer 6

Separate protein mixtures, m/z will be proportional to mass because the proteins are coated with SDS detergent which forms a micelle around the protein

Question 7

What is Size-Exclusion Chromatography?

Answer 7

Proteins can be separated on size-exclusion or using affinity tags

GST is used as an affinity tag with a linker that can be cleaved by proteases

Size exclusion:
Large molecules come out first followed by smaller molecules

Tag:

Question 8

What do proteases do?

Answer 8

Cleave proteins

Question 9

What does trypsin cleave?

Answer 9

Positive residues except pro

Question 10

What does Chymotrypsin cleave?

Answer 10

Bulky hydrophobic residues except pro