Biochem Lab Techniques

Question 1

SDS-PAGE stands for:

Answer 1

Sodium dodecyl sulfate polyacrylamide gel electrophoresis

SDS: detergent > negatively charges proteins migrate towards pos anode

Question 2

SDS page uses what type of cell with anode and cathode of what charges?

Answer 2

Electrolytic: electrical current put in
Anode is positive
Cathode is negative

Question 3

What is beta mercapto ethanol and when would it be added to a lab technique?

Answer 3

Reducing agent added to break disulfide bonds and help denature
- in gel electrophoresis

Question 4

What type of membrane is used in blotting technique?

Answer 4

Nitrocellulose membrane

Question 5

Steps of blotting procedure:

Answer 5

1. SDS - denature and apply neg charge (maybe reducing agent like beta mercapto ethanol to help break disulfide bonds)
2. Run on gel electro to separate by size
3. Contents transferred to nitrocellulose
4. Primary and secondary antibodies bind to protein of interest and

Question 6

How does ddNTP differ from deoxyribonucleotide:

Answer 6

Lack OH on 2 and 3’

Question 7

What does addition of ddNTP due to elongation?

Answer 7

premature termination, no 3’ hydroxy group for next nucleotide to attach

Question 8

What is often used in tandem with taq polymerase during PCR to stabilize charge on DNA strands?

Answer 8

Magnesium phos buffer solution, otherwise neg DNA strands would repel each other

Question 9

What is best method for transcriptional regulation?

Answer 9

RT-PCR

Question 10

Similarities of PCR and RT-PCR

Answer 10

• both use heat resistant polymerase, taq for DNA amplification
• both use primers
• both amplify DNA

Question 11

Differences between PCR and RT-PCR

Answer 11

Different templates:

• DNA for PCR
• RNA to DNA for RT PCR