4.1 test

Question 1

Transformation efficiency calculation

Answer 1

Colonies growing on plate / DNA spread onto plate (ug)

Question 2

pGLO plasmid components

Answer 2

bla: beta-lactamase, provides ampicillin resistance
GFP gene
ori
araC repressor: prevents RNA polymerase from binding to and transcribing the GFP gene

Question 3

Protein folding rules

Answer 3

1) Hydrophobic/nonpolar on the inside
2) Hydrophilic/polar on the outside
3) Acidic and basic side chains are on the protein surface and form salt bridges with each other
4) Cysteine sidechains are opposite each other -> covalent disulfide bond that stabilizes the protein

Question 4

Chromatography

Answer 4

Sample is dissolved in the mobile phase (gas/liquid) and travels through a stationary phase (a filter like silica/beads)

Question 5

Buffer order (highest to lowest salt)

Answer 5

Binding, wash, elution/TE
Hydrophilic = low salt
Hydrophobic = high salt

Question 6

Lab 1 (transformation)

Answer 6

1) Combine transformation solution (contains CaCl2 ions that neutralize negative charges) with E. Coli cells from starter plate
2) Place tube on ice
3) Immediately place it in hot water bath (95C)
4) Take it out and put it back on ice
5) Add nutrient broth to facilitate beta-lactamase expression
6) Plate the 2 solutions onto 4 different plates and incubate (+pGLO lb/amp/ara, +pGLO lb/amp, -pGLO lb, -pGLO lb/amp)

Question 7

Lab 2 - isolating GFP

Answer 7

1) Put +pGLO lb/amp/ara colony in a tube with liquid growth media and incubate to produce more GFP
2) Microcentrifuge - pour out supernatant (growth media debris) and keep pellet (only E. Coli cells)
3) Add TE solution
4) Add lysozyme
5) Freeze
6) Microcentrifuge again - keep the supernatant (proteins only) and discard pellet

Question 8

Lab 2 - purifying GFP

Answer 8

1) Put the supernatant from the previous experiment into a new microcentrifuge tube and add binding buffer
2) Snap off ends of the chromatography column, let it drain, and add equilibriation buffer to enhance beads’ hydrophobic properties
3) Pipet the supernatant (with binding buffer) through the first column
4) Transfer to 2nd column and add wash buffer
5) Transfer to 3rd column and add TE/elution buffer

Question 9

Lab 3 - electrophoresis

Answer 9

1) Transfer HIC fractions 2 and 3 to new tubes and add transformation solution, which includes SDS and tracking dye (coomassie blue)
2) Move tubes to hot water bath (95C) to denature proteins
3) Run electrophoresis
4) Use white light box
26.9 kiloDaltons