Lecture 6 Flashcards
1
Q
Filter Sterilization
A
- secondary sterilization method
- when compounds can’t be heated
2
Q
Microscopy
A
- well prepared slides and good technique make observations easier
3
Q
Resolution Equation
A
R = 0.61 (wavelength) / NA
4
Q
Spectroscopy
A
- the study of absorption and emission of radiation
5
Q
Photometry
A
- the measurement of intensity of radiation
6
Q
Spectrophotometry
A
- 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
7
Q
Photons
A
- makes up light
8
Q
higher energy
A
- shorter wavelength (blue)
9
Q
lower energy
A
- longer wavelength (red)
10
Q
light energy
A
- can generate heat or produce fluorescence
11
Q
Cuvetter
A
- a tube that holds the sample
12
Q
Transmittance
A
- the proportion of light that the sample allows to pass
- usually %
13
Q
Absorbance
A
- the amount of light the sample blocks
- calculated as
A = log (1/T)
14
Q
Blank
A
- a comparison sample with none of the measure substance
15
Q
Standard Curve
A
- a series of known samples used to generate comparison data
16
Q
General considerations of Spectrophotometry
A
- clean cuvettes: fingerprints and condensation block light
- squared cuvettes lined properly
- calibration
- adequate volume in cuvette
17
Q
Absorption Spectrum
A
- measure sample at various wavelengths
- plot curve of absorbance
- useful to determine the optimal wavelength for measuring Abs
18
Q
Beer-Lambert Law
A
- the taller the glass, the darker the brew, the less the amount of light that gets through
19
Q
Beer-Lambert Law Equation
A
A = elc
e: molar absorptivity (L/mol cm)
l: path length (cm) (usually constant)
c: concentration (mol/L)
20
Q
Direct Spectrophotometric Measurements: Bacterial Cells
A
- light scattering measured at 595-600 nm
21
Q
Direct Spectrophotometric Measurements: DNA or RNA
A
- absorb UV light at 260 nm
- Abs x dilution factor x 50 = [DNA] in µg/mL
- with standard 1cm path length
22
Q
Direct Spectrophotometric Measurements: Proteins
A
- directly absorb UV light at 280 nm (varies by conformation)
- more sensitive assays use indirect measurement
23
Q
Determining DNA Purity
A
- 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
24
Q
Pure DNA
A
- 0
- ~1.8 is considered good
25
Q
Pure Protein
A
0.55
26
Q
Indirection Protein Assays: Bradford Assay
A
- 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
