Class Notes (IMPORTANT THINGS TO KNOW)

Question 1

What is the molar ratio of insert to vector?

Answer 1

2 (insert) : 1 (vector)

Question 2

1 AU (atomic unit)

Answer 2

50 ug/mL DNA

Question 3

1 base pair of DNA

Answer 3

660 g/mol

Question 4

what are the five steps of the typical strategy for expression of a recombinant gene product?

Answer 4

1. amplify gene from source DNA by PCR
2. clone PCR product into an expression vector by ligation
3. verify clonal plasmids by restriction mapping/gel electrophoresis
4. transform expression host (electroporation/chemically induced competency)
5. grow transform cells in liquid medium

Question 5

epitope tag

Answer 5

peptide sequence for which a monoclonal antibody will bind

Question 6

what are the three steps of PCR?

Answer 6

1. denaturation
2. annealing
3. extension/amplification

Question 7

after which PCR cycle will there be 2 correct products?

Answer 7

following the 3rd PCR cycle there will be two correct products. This number will double exponentially every cycle.

Question 8

How do you approach a problem that gives you the length/absorbance of plasmid and insert

Answer 8

1. Start by doing STOICH to find the Molarity of the plasmid and insert
2. Then set up the equation
   (mols i / mols p) = (2)([I](vol. i)) / ([P](vol. of p))
3. Also do equation (vol. i) + (vol p) = total Vol.
4. Use the two equations to solve for the two unknowns

Question 9

what is the amplification factor of PCR?

Answer 9

2^(n-2) where n is the number of cycles

Question 10

The equation for Data analysis of DNA

Answer 10

log(L) = mD + b

L - length of DNA
D - migration distance (from center of well)

Question 11

Direction of Synthesis during artificial DNA synthesis

Answer 11

3’ —-> 5’

Question 12

How do you find the yield given an efficiency?

Answer 12

n^(N-1)
n - single step coupling efficiency
N - # of nucleotides in the oligonucleotide

Question 13

What are the 5 guidelines when designing a primer

Answer 13

1. Must hybridize to complementary positions matching designated sequence
2. Prefered G/C content around 40-60%
3. 50°C ≤ Tm ≤ 60°C
4. Primers must not be complementary to self or each other
5. Prefer ‘G/C clamp’ = 2-3 of last 5 nucleotides

Question 14

How do you find the melting temperature

Answer 14

(2°C)(# A/T) + (4°C)(# G/C) = Tm

Question 15

How do you find the annealing temperature

Answer 15

Tm - 5°C = Ta

Question 16

What is the typical temperature most proteins denature at

Answer 16

95°C

Question 17

What is different about a ddNTP

Answer 17

No 3’-OH group