experiment 9: UV-vis spectroscopy

Question 1

know the differences between microplate readers and single cell spectrophotometers

Answer 1

microplate reader: measures multiple samples (wells) at one specific wavelength

single cell spectrophotometer: measures on sample (cuvette) at multiple wavelengths

Question 2

know the basic and proper usage of cuvettes and spectrophotometers

Answer 2

• do not handle cuvettes on the bottom, touch near the top where the notch is
• insert cuvette so that light will pass through the side with the notch
• do not wipe cuvettes with kimwipe, can cause scratches
• avoid oil/fingerprints on the light passing surfaces
• avoid any bubbles or small particles in the sample
• spectrophotometer measures cuvette that is in the 9’ o clock position in the turret

Question 3

use the Beer-Lambert equation to determine the concentrations of biological molecules

Answer 3

A = bCE

• A: absorbance at a particular wavelength
• b: path length of cuvette, usually 1 cm
• C: concentration of the sample (M)
• E: molar extinction coefficient at a particular wavelength (1/cm*M)
• as concentration increases, absorbance increases

Question 4

analyze and interpret wavelength spectra of common biological samples

Answer 4

• bacterial growth: lag phase, growth phase, stationary phase, death phase
• changing concentration: peaks and troughs remain unchanged
• changing salt and buffer components: peaks and troughs can shift, amplitude can change (buffer)

Question 5

calculate bacterial cell numbers, protein concentration, nucleic acid concentration and enzyme rates using spectrophotometer data

Answer 5

• bacterial cell numbers: you can estimate that an OD600 of 1 corresponds to approximately 8x10^8 e.coli cells per ml
• protein concentration (in DNA/protein mixture): (1.55 x A280) - (0.76 x A260) = [protein mg/ml]
• nucleic acid concentration:
• enzyme rate:

Question 6

properly set up a blank sample

Answer 6

• blank used as a reference
• typically Tris + a salt while the sample includes DNA
• if you get a negative reading the wrong blank could have been used or placed in the wrong orientation

Question 7

typical uses of UV-vis spectroscopy

Answer 7

• monitor bacterial growth
• quantify DNA or protein concentration
• monitor enzyme kinetics
• detect conformational changes in proteins or nucleic acids
• observing ligand-binding reactions
• measures 200-760 nm

Question 8

cuvette types and uses

Answer 8

• UV quartz: measures at 190-750 nm, very expensive
• methacrylate (plastic): measures at 260-750 nm, inexpensive and disposable
• polystyrene: measures at 340-750 nm, inexpensive but limited to VIS range
• glass: measures at 340-750 nm, inexpensive but limited to VIS range