Lecture 6

Question 1

Filter Sterilization

Answer 1

• secondary sterilization method

- when compounds can’t be heated

Question 2

Microscopy

Answer 2

• well prepared slides and good technique make observations easier

Question 3

Resolution Equation

Answer 3

R = 0.61 (wavelength) / NA

Question 4

Spectroscopy

Answer 4

• the study of absorption and emission of radiation

Question 5

Photometry

Answer 5

• the measurement of intensity of radiation

Question 6

Spectrophotometry

Answer 6

• spectroscopy + photometry
• in a solution every component may interact with a light (electromagnetic radiation) source
• study of the interaction of light on a sample
• can tell what compounds may be present and in what quantities

Question 7

Photons

Answer 7

• makes up light

Question 8

higher energy

Answer 8

• shorter wavelength (blue)

Question 9

lower energy

Answer 9

• longer wavelength (red)

Question 10

light energy

Answer 10

• can generate heat or produce fluorescence

Question 11

Cuvetter

Answer 11

• a tube that holds the sample

Question 12

Transmittance

Answer 12

• the proportion of light that the sample allows to pass

- usually %

Question 13

Absorbance

Answer 13

• the amount of light the sample blocks
• calculated as
  A = log (1/T)

Question 14

Blank

Answer 14

• a comparison sample with none of the measure substance

Question 15

Standard Curve

Answer 15

• a series of known samples used to generate comparison data

Question 16

General considerations of Spectrophotometry

Answer 16

• clean cuvettes: fingerprints and condensation block light
• squared cuvettes lined properly
• calibration
• adequate volume in cuvette

Question 17

Absorption Spectrum

Answer 17

• measure sample at various wavelengths
• plot curve of absorbance
• useful to determine the optimal wavelength for measuring Abs

Question 18

Beer-Lambert Law

Answer 18

• the taller the glass, the darker the brew, the less the amount of light that gets through

Question 19

Beer-Lambert Law Equation

Answer 19

A = elc

e: molar absorptivity (L/mol cm)
l: path length (cm) (usually constant)
c: concentration (mol/L)

Question 20

Direct Spectrophotometric Measurements: Bacterial Cells

Answer 20

• light scattering measured at 595-600 nm

Question 21

Direct Spectrophotometric Measurements: DNA or RNA

Answer 21

• absorb UV light at 260 nm
• Abs x dilution factor x 50 = [DNA] in µg/mL
• with standard 1cm path length

Question 22

Direct Spectrophotometric Measurements: Proteins

Answer 22

• directly absorb UV light at 280 nm (varies by conformation)
• more sensitive assays use indirect measurement

Question 23

Determining DNA Purity

Answer 23

• DNA has a peak @ 260nm
• protein has a peak @ 280 nm
• by comparing A260/A280 ratio we get an idea of the quality of a DNA or protein isolation

Question 24

Pure DNA

Answer 24

2. 0

- ~1.8 is considered good

Question 25

Pure Protein

Answer 25

0.55

Question 26

Indirection Protein Assays: Bradford Assay

Answer 26

- measurement of colour change when Coomassie Blue dye binds protein - unbound dye Absmax=465nm (reddish brown) - bound dye Absmax=595nm (blue) - strongest interaction with basic amino acid (can vary) - upper limit for linear detection = 1 mg/mL - dye can precipitate if detergents are present