(M) L2.2 : Gel Electrophoresis Flashcards
(110 cards)
1
Q
Familiarize !
methods for electrophoresis
A
- prepare agarose gel
- pour into casting tray with comb and allow to solidify
- add running buffer, load samples and marker
- run gel at constant voltage until band separation occurs
- view DNA on UV light box and show results
2
Q
what is used to view the fluorescent DNA in the gel
A
UV transilluminator
3
Q
this causes the stained DNA bands to fluoresce
A
UVL
4
Q
what must be the same to DNA being measured to quantify the DNA
A
size and dye
5
Q
T or F
Good quality DNA:
bond is compact, with double bond or faint band
A
F (NO double bond or faint band)
6
Q
the most common reason for running a gel
A
evaluate the quality of DNA
7
Q
T or F
agarose is difficult to prepare and needs a large concentration of agar
A
F (easy and low concentration)
8
Q
T or F
Agar resolution is better than filter paper
A
T
9
Q
T or F
Small quantities of proteins can be separated and recovered in agarose gel electrophoresis
A
F ( large quantities, polyacrylamide yung small amount)
10
Q
T or F
Adsorption of negatively charged protein molecule is negligible in agarose gel electrophoresis
A
T
11
Q
T or F
agarose gel electrophoresis adsorbs proteins relatively less compared to other mediums
A
T
12
Q
T or F
Agarose gel has better resolution compared to polyacrylamide gel
A
F
13
Q
T or F
different sources and batches of agar tend to give different results and purification is often necessary
A
T
14
Q
Agarose or PAG?
Polysaccharide extracted from seaweed
A
agarose
15
Q
Agarose or PAG?
cross-linked polymer of acrylamide
A
PAG
16
Q
Agarose or PAG?
gel casted horizontally
A
agarose
17
Q
Agarose or PAG?
gel casted vertically
A
PAG
18
Q
Agarose or PAG?
Separates large molecules
A
agarose
19
Q
Agarose or PAG?
separates small molecules
A
PAG
20
Q
Agarose or PAG?
Commonly used for DNA separation
A
agarose
21
Q
Agarose or PAG?
used for DNA or protein separations
A
PAG
22
Q
Agarose or PAG?
staining can be done before or during the gel pour
A
agarose
23
Q
Agarose or PAG?
staining can be done after pouring the gel
A
PAG
24
Q
how is the molecular sized of nucleic acid expressed?
A
molecular weight
25
molecular weight of a nucleic acid is equivalent to?
number of bps in the molecule
26
T or F
small fragments move slowly across the gel
F (faster and farther)
27
T or F
large fragments moves slowly due to greater frictional drag
T
28
how does the molecules move through the gel
reptation (snake like movement)
29
T or F
The concentration of agarose is inversely proportional to DNA size
T
30
what determines the pore size
agarose concentration
31
what size of DNA does high agarose concentration facilitate the separation of?
smaller DNA (0.1 to 2kb)
32
what size of DNA does low agarose concentration facilitate the separation of?
larger DNA (5-60kb)
33
T or F
supercoiled circular DNA, relaxed circular DNA and linear DNA of the same molecular weight will migrate at different rates through the gel
T
34
arrange the following from the LAST to the FIRST to migrate through the gel
supercoiled DNA
circular DNA
linear double stranded DNA
1. circular DNA
2. linear
3. supercoiled
35
why does supercoiled DNA migrate first, farther, and faster?
it's more compact and has lesser frictional resistance
36
what happens if a supercoiled DNA is broken due to nick / cut
reverts to circular DNA
37
what happens if circular DNA is cut?
it will become linear double stranded DNA
38
At what speed does linear double stranded DNA migrate?
normal speed
39
what form does bacterial chromosomal DNA take?
closed circular double helix
40
what is the native conformation found in vivo, occurs when extra twists are introduced to the double helix
form I - supercoiled circular DNA
41
42
The slowest migrating form in an agarose gel, nicked by topoisomerases which allows the polymerases to gain access to DNA
form II - nicked circular DNA
43
occurs when the DNA helix is cut in both strands at the same place
Form III - linear DNA
44
Rate of migration is _______ to the voltage applied
proportional
45
T or F
The higher the voltage, the faster the rate of migration
T
46
what happens if the voltage is too high?
generates heat that may denature the DNA and melt the gel
47
low or high voltage?
sharper resolution in larger DNA fragments and better visualization of bands
low
48
low or high voltage?
shorter run time
high
49
low or high voltage?
DNA may diffuse
low voltage
50
what is the optimal voltage for max resolution of DNA fragments less than 2kb in size
5-8 V/cm
51
what component of electrophoresis buffer affects DNA mobility
(2)
composition and ionic strength
52
why does DNA migrate slowly in water ?
electrical conductivity is minimal
53
what is the disadvantage of using high ionic strength (10x buffer)
generates significant heat -> gel melts and DNA denatures
54
what are the two standard buffers?
Tris borate EDTA (TBE)
Tris acetate EDTA (TAE)
55
What type of agents are dyes usually?
intercalating agents
56
type of agarose that has a high melting temperature?
standard
57
what is the gelling and melting temp of standard agarose?
gels at 35 to 38 degrees Celsius
melts at 90-95 degrees Celsius
58
from what two species of seaweeds is standard agarose derived from
gelidium and glacilaria
59
what is the gelling an melting temperature of low melting temp agarose
gels at 35 degrees Celsius
melts at 65 degrees Celsius
60
Troubleshooting
- center of the gel running hotter than both ends
- power conditions are excessive
smile effect band pattern curves upward at both sides of the gels
61
Troubleshooting
sample overload
curved bans, smiles
62
Troubleshooting
- agarose improper
- salt concentration is too high
- excessive power and heating
- sample spilled out of the well
- incomplete digest nuclease contamination (bad enzyme)
band smearing and streaking
63
Troubleshooting
voltage gradient is too high
gels crack
64
Troubleshooting
- sample overload
- sample precipitation
vertical streaking bands
65
Troubleshooting
- poor polymerization around sample wells
- salts in sample
distorted band
66
Troubleshooting
- running buffer is too concentrated
- excessive salt in the sample
- runs too fast
run taking an unusually long time
67
Troubleshooting
- running buffer is too diluted
- voltage is too high
poor resolution
68
electrophoresis that uses more than one electrical field, separates very large DNA molecules
pulse-field gel electrophoresis
69
what directions can pulse-field gel electrophoresis go?
one direction (horizontal or vertical)
opposite directions
different angles
70
why is the mobility of DNA molecules dependent on the electrical field applied to the gel?
it disrupts hydrogen bonds which reorientates agarose fibers and fiber bundles
71
what does the reorientation of agarose fibers and fiber bundles produce?
larger pore size
72
Familiarize !
study the process of PFGE
go na bhie
73
what does PFGE use as molecular scissors
restriction enzymes
74
what takes place due to the application of electrical field that constantly changes in direction
separation of sizes
75
T or F
per lane, only one band may be seen under UV light
F (multiple bands)
76
what is the main difference between the types of PFGE
direction of electrical field
77
What type of Pulse-Field Gel electrophoresis is this?
contour clamped homogenous electric fields
78
how many passive electrodes are in contour-clamped homogenous electric field (CHEF) and how are they arranged?
24 passive electrodes, arranged hexagonally
79
why are the passive electrodes arranged hexagonally for CHEF?
to create distortion on the edge of the chamber
80
can the electrodes regulate the voltage in a unit electric field (CHEF)
yes
81
are these precisely controlled in CHEF? (yes or no)
size, shape, location, coordination, stability, and continuity
no (all except shape are precisely controlled)
82
What type of Pulse-Field Gel electrophoresis is this?
Field-inversion Gel electrophoresis
83
why are the two fields in FIGE arranged in separate straight angles?
so that the electric field direction can be reversed periodically
84
What does shorter time pulses in FIGE do for the molecules?
molecules moves forward
85
What does longer time pulses in FIGE do for the molecules?
molecules moves reversely
86
what problem does FIGE solve ?
comigration of nucleic acids and protein detergent complexes
87
this is common when both nucleic acids and protein detergent complexes are larger than a threshold size
comigration
88
FIGE provides good resolution over ______
800 kbp
89
modification of Field-inversion gel electrophoresis
Asymmetric Field-inversion Gel electrophoresis (AFIGE)
90
what is AFIGE used for?
detection of DNA double-strand breaks (DSBs)
91
what can you measure in an inverted and asymmetric electric field quantitatively
rate of DNA breakage
92
What type of Pulse-Field Gel electrophoresis is this?
Orthogonal-Field Alternation Gel Electrophoresis (OFAGE)
93
Orthogonal-Field alternation gel electrophoresis is a _______ electrophoresis system
vertical
94
what does the two orthogonal electric fields produce when alternately supplied to the agarose gel
non linear and dissimilar electric fields
95
what is the size range for DNA molecules that can be separated by OFAGE?
1000 kb and 2000 kb
96
What type of Pulse-Field Gel electrophoresis is this?
Rotating gel electrophoresis
97
in this method, the agarose gel is rotated between two angles periodically and the power supply is turned off after switching the angle during electrophoresis
rotating gel electrophoresis (RGE)
98
rotating gel electrophoresis is suitable for separating DNA with _____
50kb to 6000 kb
99
this method of PFGE, 24 electrodes are arranged in a close contour
Programmable autonomously controlled electrodes
100
this system can produce an unlimited number of controlled homogenous electric fields, voltage gradients and direction and duration flow
PACE system
101
how can PACE electrophoresis system control all the parameters of the electric field?
independent adjustment of voltage on electrodes
102
A flexible type of PFGE, preferable to the other alternating electrophoresis methods
PACE system
103
what size DNA fragments are separable in PACE system?
100 bp to more than 6Mb
104
familiarize the applications of PFGE
1. Genotyping or genetic fingerprinting
2. It is the gold standard in epidemiological studies of pathogenic organisms
3. monitors changes in bacteria
4. shows relationship of different strains of a single species
105
type of electrophoresis carried out in a fused-silica capillary tube
capillary gel electrophoresis
106
what type of voltage is applied in capillary gel electrophoresis
high voltage
107
where do you acquire data in capillary gel electrophoresis?
detector window
108
T or F
Capillary gel electrophoresis has higher resolution, greater speed, online detection, minimal use of samples and buffer
T
109
how many samples can be processed at a time by a single capillary?
1 sample for each capillary
110
T or F
capillaries are fragile
T
