Class Notes (IMPORTANT THINGS TO KNOW) Flashcards
What is the molar ratio of insert to vector?
2 (insert) : 1 (vector)
1 AU (atomic unit)
50 ug/mL DNA
1 base pair of DNA
660 g/mol
what are the five steps of the typical strategy for expression of a recombinant gene product?
- amplify gene from source DNA by PCR
- clone PCR product into an expression vector by ligation
- verify clonal plasmids by restriction mapping/gel electrophoresis
- transform expression host (electroporation/chemically induced competency)
- grow transform cells in liquid medium
epitope tag
peptide sequence for which a monoclonal antibody will bind
what are the three steps of PCR?
- denaturation
- annealing
- extension/amplification
after which PCR cycle will there be 2 correct products?
following the 3rd PCR cycle there will be two correct products. This number will double exponentially every cycle.
How do you approach a problem that gives you the length/absorbance of plasmid and insert
- Start by doing STOICH to find the Molarity of the plasmid and insert
- Then set up the equation
(mols i / mols p) = (2)([I](vol. i)) / ([P](vol. of p)) - Also do equation (vol. i) + (vol p) = total Vol.
- Use the two equations to solve for the two unknowns
what is the amplification factor of PCR?
2^(n-2) where n is the number of cycles
The equation for Data analysis of DNA
log(L) = mD + b
L - length of DNA
D - migration distance (from center of well)
Direction of Synthesis during artificial DNA synthesis
3’ —-> 5’
How do you find the yield given an efficiency?
n^(N-1)
n - single step coupling efficiency
N - # of nucleotides in the oligonucleotide
What are the 5 guidelines when designing a primer
- Must hybridize to complementary positions matching designated sequence
- Prefered G/C content around 40-60%
- 50°C ≤ Tm ≤ 60°C
- Primers must not be complementary to self or each other
- Prefer ‘G/C clamp’ = 2-3 of last 5 nucleotides
How do you find the melting temperature
(2°C)(# A/T) + (4°C)(# G/C) = Tm
How do you find the annealing temperature
Tm - 5°C = Ta
