Media Mayhem
How many plates can be made from 500 mL of LB agar?
20 plates
What chemical and concentration is used to disinfect surfaces in the biosafety cabinet?
70% ethanol
What temperature is used for heat shock during transformation?
42°C
What detergent is present in the lysis buffer?
Triton X-100
What is the molecular weight of the HIV-p17?
16254.29 g/mol
If you are making a 100 ml stock solution of the premixed LB powder, how much LB powder should you use?
2.0 g (bottle says 20 g for 1 liter)
What should never be blocked inside the biosafety cabinet?
Air grille
How long do cells stay on ice before heat shock?
30 minutes
What temperature should centrifugation occur at during lysis?
4°C
How much sample is loaded into each well?
13-15 µL
What pH should minimal media be adjusted to?
pH 7.00–7.05
Your LB stock solution has turned a bit cloudy, what should you do?
assume it is contaminated, discard, and make a new stock of it
How long is the heat shock step?
30 seconds
What type of resin is used for purification?
Ni-NTA resin
What is the extinction coefficient for HIV-p17?
17085 M-1cm-1
What are the three ingredients needed to make LB media from scratch?
Tryptone, yeast extract, & Sodium chloride
Why are thiamine, biotin, and ampicillin sterile filtered instead of autoclaved?
They are heat sensitive
How much plasmid is added to competent cells?
1 µL
Which buffer has the second highest imidazole concentration?
Equilibration buffer (50 mM)
What voltage is used after the first 30 minutes in the electrophoresis step?
100V
What is the molecular weight of the ampicillin we use and what temperature do we store it at?
371.4 g/mol & -20 'C
What autoclave cycle should be used for liquid media?
Liquid 30 or 60
How much of the lysate do you save to be used for the gel?
100 uL
Why does imidazole help elute His-tagged protein?
it competes with histidine residues for nickel binding
Why are SDS and BME added to protein samples before running the gel?
SDS denatures proteins + gives uniform negative charge & BME breaks disulfide bonds