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Mol Bio Lab > (M) L2.2 : Gel Electrophoresis > Flashcards

(M) L2.2 : Gel Electrophoresis Flashcards

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1
Q

Familiarize !

methods for electrophoresis

A
  1. prepare agarose gel
  2. pour into casting tray with comb and allow to solidify
  3. add running buffer, load samples and marker
  4. run gel at constant voltage until band separation occurs
  5. view DNA on UV light box and show results
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2
Q

what is used to view the fluorescent DNA in the gel

A

UV transilluminator

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3
Q

this causes the stained DNA bands to fluoresce

A

UVL

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4
Q

what must be the same to DNA being measured to quantify the DNA

A

size and dye

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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)

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6
Q

the most common reason for running a gel

A

evaluate the quality of DNA

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7
Q

T or F

agarose is difficult to prepare and needs a large concentration of agar

A

F (easy and low concentration)

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8
Q

T or F

Agar resolution is better than filter paper

A

T

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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)

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10
Q

T or F

Adsorption of negatively charged protein molecule is negligible in agarose gel electrophoresis

A

T

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11
Q

T or F

agarose gel electrophoresis adsorbs proteins relatively less compared to other mediums

A

T

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12
Q

T or F

Agarose gel has better resolution compared to polyacrylamide gel

A

F

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13
Q

T or F

different sources and batches of agar tend to give different results and purification is often necessary

A

T

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14
Q

Agarose or PAG?

Polysaccharide extracted from seaweed

A

agarose

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15
Q

Agarose or PAG?

cross-linked polymer of acrylamide

A

PAG

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16
Q

Agarose or PAG?

gel casted horizontally

A

agarose

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17
Q

Agarose or PAG?

gel casted vertically

A

PAG

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18
Q

Agarose or PAG?

Separates large molecules

A

agarose

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19
Q

Agarose or PAG?

separates small molecules

A

PAG

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20
Q

Agarose or PAG?

Commonly used for DNA separation

A

agarose

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21
Q

Agarose or PAG?

used for DNA or protein separations

A

PAG

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22
Q

Agarose or PAG?

staining can be done before or during the gel pour

A

agarose

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23
Q

Agarose or PAG?

staining can be done after pouring the gel

A

PAG

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24
Q

how is the molecular sized of nucleic acid expressed?

A

molecular weight

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25
Q

molecular weight of a nucleic acid is equivalent to?

A

number of bps in the molecule

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26
Q

T or F

small fragments move slowly across the gel

A

F (faster and farther)

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27
Q

T or F

large fragments moves slowly due to greater frictional drag

A

T

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28
Q

how does the molecules move through the gel

A

reptation (snake like movement)

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29
Q

T or F

The concentration of agarose is inversely proportional to DNA size

A

T

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30
Q

what determines the pore size

A

agarose concentration

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31
Q

what size of DNA does high agarose concentration facilitate the separation of?

A

smaller DNA (0.1 to 2kb)

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32
Q

what size of DNA does low agarose concentration facilitate the separation of?

A

larger DNA (5-60kb)

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33
Q

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

A

T

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34
Q

arrange the following from the LAST to the FIRST to migrate through the gel

supercoiled DNA
circular DNA
linear double stranded DNA

A
  1. circular DNA
  2. linear
  3. supercoiled
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35
Q

why does supercoiled DNA migrate first, farther, and faster?

A

it’s more compact and has lesser frictional resistance

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36
Q

what happens if a supercoiled DNA is broken due to nick / cut

A

reverts to circular DNA

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37
Q

what happens if circular DNA is cut?

A

it will become linear double stranded DNA

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38
Q

At what speed does linear double stranded DNA migrate?

A

normal speed

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39
Q

what form does bacterial chromosomal DNA take?

A

closed circular double helix

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40
Q

what is the native conformation found in vivo, occurs when extra twists are introduced to the double helix

A

form I - supercoiled circular DNA

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41
Q
A
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42
Q

The slowest migrating form in an agarose gel, nicked by topoisomerases which allows the polymerases to gain access to DNA

A

form II - nicked circular DNA

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43
Q

occurs when the DNA helix is cut in both strands at the same place

A

Form III - linear DNA

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44
Q

Rate of migration is _______ to the voltage applied

A

proportional

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45
Q

T or F

The higher the voltage, the faster the rate of migration

A

T

46
Q

what happens if the voltage is too high?

A

generates heat that may denature the DNA and melt the gel

47
Q

low or high voltage?

sharper resolution in larger DNA fragments and better visualization of bands

A

low

48
Q

low or high voltage?

shorter run time

A

high

49
Q

low or high voltage?

DNA may diffuse

A

low voltage

50
Q

what is the optimal voltage for max resolution of DNA fragments less than 2kb in size

A

5-8 V/cm

51
Q

what component of electrophoresis buffer affects DNA mobility

(2)

A

composition and ionic strength

52
Q

why does DNA migrate slowly in water ?

A

electrical conductivity is minimal

53
Q

what is the disadvantage of using high ionic strength (10x buffer)

A

generates significant heat -> gel melts and DNA denatures

54
Q

what are the two standard buffers?

A

Tris borate EDTA (TBE)
Tris acetate EDTA (TAE)

55
Q

What type of agents are dyes usually?

A

intercalating agents

56
Q

type of agarose that has a high melting temperature?

A

standard

57
Q

what is the gelling and melting temp of standard agarose?

A

gels at 35 to 38 degrees Celsius
melts at 90-95 degrees Celsius

58
Q

from what two species of seaweeds is standard agarose derived from

A

gelidium and glacilaria

59
Q

what is the gelling an melting temperature of low melting temp agarose

A

gels at 35 degrees Celsius
melts at 65 degrees Celsius

60
Q

Troubleshooting

  • center of the gel running hotter than both ends
  • power conditions are excessive
A

smile effect band pattern curves upward at both sides of the gels

61
Q

Troubleshooting

sample overload

A

curved bans, smiles

62
Q

Troubleshooting
- agarose improper
- salt concentration is too high
- excessive power and heating
- sample spilled out of the well
- incomplete digest nuclease contamination (bad enzyme)

A

band smearing and streaking

63
Q

Troubleshooting

voltage gradient is too high

A

gels crack

64
Q

Troubleshooting

  • sample overload
  • sample precipitation
A

vertical streaking bands

65
Q

Troubleshooting
- poor polymerization around sample wells
- salts in sample

A

distorted band

66
Q

Troubleshooting

  • running buffer is too concentrated
  • excessive salt in the sample
  • runs too fast
A

run taking an unusually long time

67
Q

Troubleshooting

  • running buffer is too diluted
  • voltage is too high
A

poor resolution

68
Q

electrophoresis that uses more than one electrical field, separates very large DNA molecules

A

pulse-field gel electrophoresis

69
Q

what directions can pulse-field gel electrophoresis go?

A

one direction (horizontal or vertical)
opposite directions
different angles

70
Q

why is the mobility of DNA molecules dependent on the electrical field applied to the gel?

A

it disrupts hydrogen bonds which reorientates agarose fibers and fiber bundles

71
Q

what does the reorientation of agarose fibers and fiber bundles produce?

A

larger pore size

72
Q

Familiarize !

study the process of PFGE

A

go na bhie

73
Q

what does PFGE use as molecular scissors

A

restriction enzymes

74
Q

what takes place due to the application of electrical field that constantly changes in direction

A

separation of sizes

75
Q

T or F

per lane, only one band may be seen under UV light

A

F (multiple bands)

76
Q

what is the main difference between the types of PFGE

A

direction of electrical field

77
Q

What type of Pulse-Field Gel electrophoresis is this?

A

contour clamped homogenous electric fields

78
Q

how many passive electrodes are in contour-clamped homogenous electric field (CHEF) and how are they arranged?

A

24 passive electrodes, arranged hexagonally

79
Q

why are the passive electrodes arranged hexagonally for CHEF?

A

to create distortion on the edge of the chamber

80
Q

can the electrodes regulate the voltage in a unit electric field (CHEF)

A

yes

81
Q

are these precisely controlled in CHEF? (yes or no)

size, shape, location, coordination, stability, and continuity

A

no (all except shape are precisely controlled)

82
Q

What type of Pulse-Field Gel electrophoresis is this?

A

Field-inversion Gel electrophoresis

83
Q

why are the two fields in FIGE arranged in separate straight angles?

A

so that the electric field direction can be reversed periodically

84
Q

What does shorter time pulses in FIGE do for the molecules?

A

molecules moves forward

85
Q

What does longer time pulses in FIGE do for the molecules?

A

molecules moves reversely

86
Q

what problem does FIGE solve ?

A

comigration of nucleic acids and protein detergent complexes

87
Q

this is common when both nucleic acids and protein detergent complexes are larger than a threshold size

A

comigration

88
Q

FIGE provides good resolution over ______

A

800 kbp

89
Q

modification of Field-inversion gel electrophoresis

A

Asymmetric Field-inversion Gel electrophoresis (AFIGE)

90
Q

what is AFIGE used for?

A

detection of DNA double-strand breaks (DSBs)

91
Q

what can you measure in an inverted and asymmetric electric field quantitatively

A

rate of DNA breakage

92
Q

What type of Pulse-Field Gel electrophoresis is this?

A

Orthogonal-Field Alternation Gel Electrophoresis (OFAGE)

93
Q

Orthogonal-Field alternation gel electrophoresis is a _______ electrophoresis system

A

vertical

94
Q

what does the two orthogonal electric fields produce when alternately supplied to the agarose gel

A

non linear and dissimilar electric fields

95
Q

what is the size range for DNA molecules that can be separated by OFAGE?

A

1000 kb and 2000 kb

96
Q

What type of Pulse-Field Gel electrophoresis is this?

A

Rotating gel electrophoresis

97
Q

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

A

rotating gel electrophoresis (RGE)

98
Q

rotating gel electrophoresis is suitable for separating DNA with _____

A

50kb to 6000 kb

99
Q

this method of PFGE, 24 electrodes are arranged in a close contour

A

Programmable autonomously controlled electrodes

100
Q

this system can produce an unlimited number of controlled homogenous electric fields, voltage gradients and direction and duration flow

A

PACE system

101
Q

how can PACE electrophoresis system control all the parameters of the electric field?

A

independent adjustment of voltage on electrodes

102
Q

A flexible type of PFGE, preferable to the other alternating electrophoresis methods

A

PACE system

103
Q

what size DNA fragments are separable in PACE system?

A

100 bp to more than 6Mb

104
Q

familiarize the applications of PFGE

A
  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
Q

type of electrophoresis carried out in a fused-silica capillary tube

A

capillary gel electrophoresis

106
Q

what type of voltage is applied in capillary gel electrophoresis

A

high voltage

107
Q

where do you acquire data in capillary gel electrophoresis?

A

detector window

108
Q

T or F

Capillary gel electrophoresis has higher resolution, greater speed, online detection, minimal use of samples and buffer

A

T

109
Q

how many samples can be processed at a time by a single capillary?

A

1 sample for each capillary

110
Q

T or F

capillaries are fragile

A

T

Mol Bio Lab (12 decks)

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