Molecular Analytical Methods

Question 1

What does column chromotography do?

Answer 1

Separate proteins

Question 2

What does SDS do to separate proteins?

Answer 2

Makes sample negatively charged
unfolds protein (solubilizes)

Question 3

What does BME do? (B-mercaptoethanol)

Answer 3

Reducing agent that breaks disulfide bonds

Question 4

If you have a protein with subunits A and B joined by disulfide bridge, what do you do before gel?

Answer 4

Heat with SDS/BME
Put on gel
Protein subunits go down gel because they’re - charged

Question 5

How do you purify gels before you load them?

Answer 5

Chromatography

Question 6

What does 2-dimensional PAGE separate by?

Answer 6

Charge and size

Question 7

How do you separate by size?

Answer 7

SDS migration

Question 8

How do you separate by charge?

Answer 8

pH gradient

Question 9

What’s the isoelectric point of a pH gel?

Answer 9

Protein has no net charge (pH neutral), so doesn’t migrate anymore

Question 10

How might you use 2-D gels to detect mutations?

Answer 10

Separate by amino acid, and then compare two gels to see if one amino acid mutated (moved on the gel)

Question 11

Why do you transfer on to a western blot?

Answer 11

Way easier to work with than gel

Question 12

What are the two antibodies for in gels?

Answer 12

They grab only specific proteins that are on your gel

Question 13

What do you add after second antibody?

Answer 13

Reagent

Question 14

On final gel - what do darker lines mean?

Answer 14

There’s more protein there

Question 15

How can you use western blot in relation to time?

Answer 15

Measure protein presence during hours after irradiation

Question 16

How does mass spec work?

Answer 16

Separate sample based on m/z

Question 17

What’s the difference between ‘identities’ and ‘positives’ in BLAST?

Answer 17

‘identities’ are exactly the same amino acid

‘positives’ are similar amino acids

Question 18

What’s SDS-PAGE useful for?

Answer 18

Separate proteins based on size

Question 19

What’s western blot useful for?

Answer 19

Visualizing proteins after you’ve used SDS-PAGE,

Question 20

What’s western blot useful for?

Answer 20

Visualizing proteins after you’ve used SDS-PAGE, you only see a few because of antibodies

Question 21

What do restriction enzymes do?

Answer 21

Cut DNA at specific places

Question 22

Why do you use EtBr on gels?

Answer 22

Binds DNA, fluoresces when you shine UV light on it

Question 23

Difference between polyacrylamide, pulsed field gel, and agarose gel?

Answer 23

Agarose =

polyacrylamide = get DNA sequence, 10-50 nucleotides in a gel

pulsed field = separation of entire chromosomes, millions of nucleotides in a gel

Question 24

Why do you add 32P-labeled nucleotides?

Answer 24

They’re radioactive, so you can detect them. Kinda like PET. Could fluoresce too

Question 25

What’s in situ hybridization?

Answer 25

Visualize genes on chromosomes (fluorescence)

Question 26

How do you stick genes in bacteria? (general)

Answer 26

Stick DNA fragment into plasmid

Question 27

What proteins do you use to stick genes in bacteria?

Answer 27

Cleave with restriction nuclease > link with ligase + atp

Question 28

What is reverse transcription for?

Answer 28

Make DNA from RNA (cDNA)

Question 29

What’s the poly-T primer do? What’s it for?

Answer 29

Anneals to poly-A tail. It’s a primer for reverse transcriptase

Question 30

What do reverse transcripases do?

Answer 30

They’re polymerases that make DNA from RNA. Still build 5’-3’

Question 31

After reverse transcriptase finishes, what two strands do you have?

Answer 31

DNA/RNA

Question 32

How do you make DNA/DNA strand instead of DNA/RNA after reverse transc?

Answer 32

Use DNA polymerase, RNA as primer

Question 33

Difference between genomic library and cDNA library?

Answer 33

cDNA doesn’t have introns!

Representation in cDNA library based on amount of RNA