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SECOND SEM 3rd YEAR > MOL BIO LAB L8 (Semis- Electrophoresis) > Flashcards

MOL BIO LAB L8 (Semis- Electrophoresis) Flashcards

1
Q

The movement of molecules by (DNA or RNA) and by (applying specific voltage) an electric current

A

Electrophoresis

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2
Q

This can occur in air or solution or in a matrix to limit migration and contain the migrating material

A

Electrophoresis

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3
Q

Each phosphate group on a nucleic acid polymer is ________ making the molecule negatively charged

A

ionized

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4
Q

Under an electric current, DNA and RNA will migrate toward?

A

the positive pole (anode)

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5
Q

In a matrix of these gels, migration under the pull of the current is impeded, depending on the size of the molecules and the spaces in the gel matrix

A

Agarose and Polyacrylamide

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6
Q

Provide resistance to the movement of molecules under the force of an electric current

A

Gel system

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6
Q

Serve as a support medium for analysis of the separated components

A

Gel or Gel system

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6
Q

Enumerate the Criteria for Best Matrix

A
  • Unaffected by electrophoresis
  • Simple to prepare
  • Amenable to modification
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7
Q

Prevent diffusion and reduce convection currents so that the separated molecules form a defined group, or “band”.

A

Gel system

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8
Q

Polymers that meet the criteria for best matrix

A

Agarose gel and Polyacrylamide

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9
Q

Q1: Small pieces of DNA (________ [bp]) are resolved on more concentrated agarose gels

Q2: How many percent of agarose gel is applied for small pieces of DNA?

A

Q1: more; 50 to 500 base pairs

Q2: 2% to 3%

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10
Q

Q1: Larger fragments of DNA (____ to _____) are best resolved
in _____ agarose concentrations.

Q2: How many percent of agarose gel is applied for larger fragments of DNA?

A

Q1: 2,000 to 50,000; lower
Q2: 0.5% to 1%.

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11
Q

Reasons why gel strength of any concentration of agarose degrades.

A
  • Decrease over time
  • Exposure to chaotropic agents such as urea
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12
Q

Agarose concentrations above 5% and below 0.5% are not practical. Because high concentration agarose will __________, whereas very low concentrations produce a _________ that is easily broken

A

impede migration; weak gel

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13
Q

Q1: Used for very large pieces of DNA and bacterial typing for epidemiological purposes

Q2: How many base pairs?

A

Q1: Pulse- Field Gel Electrophoresis

Q2: 50,000 to 250,000 base pairs

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14
Q

Enumerate all the examples of pulse-field gel configurations

A
  1. Field- inversion gel electrophoresis (FIGE)
  2. Transverse alternating-field electrophoresis (TAFE)
  3. Rotating gel electrophoresis (RGE)
  4. Contour- clamped homogeneous electric field (CHEF)
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15
Q

For these very large DNA molecules, pulses of current applied to the gel in alternating dimensions enhance migration. This process is called?

A

Pulsed-field Gel Electrophoresis (PFGE)

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16
Q

Works by alternating the positive and negative electrodes during electrophoresis

A

Field- inversion gel electrophoresis (FIGE)

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17
Q

The simplest approach to this method (PFGE)

A

Field- inversion gel electrophoresis (FIGE)

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18
Q

In this type of separation, the DNA goes periodically forward and backward

A

Field- inversion gel electrophoresis (FIGE)

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19
Q

Used for applications that require the resolution of chromosome-sized fragments of DNA, such as in bacterial typing for epidemiological purposes.

A

Alternating-field electrophoresis

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20
Q

This will yield a set of fragments that produce a band pattern specific to each type of organism

A

Enzymatic digestion of genomic DNA

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21
Q

Requires a catalyst. Has higher resolution capacity for smaller fragments

A

Polyacrylamide Gels

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22
Q

Acrylamide in combination with the cross-linker ____________, polymerizes into a matrix that has consistent resolution characteristics

A

methylene bisarcylamide

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23
Q

Natural polymer from living organisms

A

Agarose

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24
Q

Why does the Polyacrylamide gel have precise control of the polymer properties and higher resolution than can be achieved with agarose?

A

Because it is a synthetic material

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25
Q

Polymerizes upon cooling

A

Agarose gel

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26
Q

Catalysts of polyacrylamide gel can be?

A

Nucleating agents

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27
Q

The two catalysts of Polyacrylamide gel

A
  • Ammonium persulfate (APS)
  • N, N, N’, N’ - tetramethylethylenediamine (TEMED) or light activation
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28
Q

Produces free oxygen radicals in the presence of TEMED to drive the polymerization mechanism

A

Ammonium persulfate (APS)

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29
Q

If light activation is used, free radicals are generated by a photochemical process using?

A

Riboflavin plus TEMED

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30
Q

In light activation, this inhibits the polymerization process

A

Excess oxygen

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31
Q

In light activation, this is done before the addition of the nucleating agents.

A

De-aeration or removal of air

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32
Q

The main advantage of polyacrylamide over
agarose

A

higher resolution capability of polyacrylamide for small fragments.

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33
Q

With single-base resolution, polyacrylamide gels are used for:

A
  • nucleic acid sequencing
  • mutation analyses
  • nuclease protection assays
  • other applications requiring high resolution of nucleic acids
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34
Q

Separates particles by size and charge

A

Capillary electrophoresis

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35
Q

In Capillary Electrophoresis, if the size is small, it migrates ______, whereas if it is large, it migrates _____.

A

faster; slower

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36
Q

In Capillary Electrophoresis, if it is negatively charge, it migrates _____, whereas if it is positively charged, it migrates _____.

A

fast; slow

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37
Q

__________ charged molecules are completely ionized at high pH whereas
___________ charged solutes are completely protonated in low-pH buffers

A

negatively; positively

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38
Q

To simplify:
Small and negatively charge particles- fast migration

Large and positively charge particles- slow migration

A
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39
Q

Give the pH: Low or High
1. Negatively charged
2. Positively charged

A
  1. High pH
  2. Low pH
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40
Q

Identify:
- Size and charge (charge/mass ratio)
- Increased sensitivity and immediate detection

A

Capillary Electrophoresis

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41
Q

These do not separate well in solution.

A

Nucleic acids (DNA & RNA)

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42
Q

Introducing a _____ inside the capillary establishes resolution by impeding nucleic acid migration according to size more than charge

A

polymer

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43
Q

In capillary electrophoresis, It is important that the nucleic acid be _________ so that it will be separated according to its size because the secondary structure will affect the migration speed

A

completely denatured (single stranded)

44
Q

Advantages of Capillary system

A

increased sensitivity
and immediate detection

45
Q

Carry the current and protect the samples during electrophoresis

A

Buffer system

46
Q

Buffer most commonly used for DNA

A

Tris-Buffer

47
Q

Types of Tris-Buffers

A
  1. Tris-borate EDTA (TBE)
  2. Tris-phosphate EDTA (TPE)
  3. Tris-acetate EDTA (TAE)
48
Q

Has a greater buffering capacity than TAE.

A

Tris-borate EDTA (TBE)

49
Q

Ion species are more easily exhausted in this Tri-buffer during extended or high-voltage electrophoresis

A

Tris- acetate EDTA (TAE)

50
Q

DNA will migrate twice as fast in ____ than in _____ in a constant current

A

TAE; TBE

51
Q

Modify sample molecules in ways that affect their migration

A

Buffer Additives

52
Q

Examples of Buffer Additives

A
  1. Formamide
  2. Urea
  3. Detergents
53
Q

Break hydrogen bonds between complementary strands or within the small strand of DNA or RNA

A

Denaturing agents (Formamide & Urea)

54
Q

These gel systems maintain this conformation such that intrachain hybridization (folding) of the nucleic acid molecules does not affect migration speeds, and separation occurs strictly according to the size or length of
the molecule.

A

Urea and heat in the gel systems

55
Q

Hinder complementary
sequences from reannealing

A

Formamide and heat

56
Q

Reacts with amino groups on the RNA to prevent base pairing between complementary nucleotides and with aldehyde

A

Denaturing Agents (RNA)
methylmercuric hydroxide (MMH)

57
Q

These are run in acrylic gel boxes or baths that are divided into two parts, with a platform

A

Horizontal gels

58
Q

The thickness of the gel and volume of the buffer affect the _____, therefore the migration of the sample.

A

Current

59
Q

This fills both compartments and makes a continuous system through which the current flows, this is also where the gel is submerged

A

Electrophoresis buffer

60
Q

These parameters are kept constant for consistent results

A

thickness of the gel and volume of the buffer

61
Q

Horizontal gels are sometimes referred to as ?

A

submarine gels

62
Q

Volume of the gel solution will determine the ________ of the gel

A

thickness

63
Q

Agarose is mixed at a certain percentage (w/v) with electrophoresis buffer and heated on a ______ or by _______to dissolve and melt the agarose

A

heat block; microwave

64
Q

Molten agarose is cooled to between?

A

55C and 65C

65
Q

Inserted into the top of the gel to create holes, or wells, in the gel into which the sample will be loaded

A

Comb

66
Q

Statement 1: The size of the teeth in the comb will determine the _______ of the well for the sample,

Stament 2: The number of teeth in the comb will determine the __________ that are available in the gel to receive the samples

A

Statement 1: capacity

Statement 2: number of wells

67
Q

2 Types of Combs

A
  1. Regular combs- horizontal gel
  2. Sharkstooth combs - vertical gel
68
Q

These are cast between glass plates that are separated by spacers.

A

Vertical gels

69
Q

The spacers determine the thickness of the gel, ranging from ____ to ____

A

0.05 to 4 mm

70
Q

The bottom of the vertical gel is secured by ____ or by a _____ in specially designed gel casting trays.

A

tape; gasket

71
Q

After the addition of catalyst and nucleating agents, the liquid acrylamide is poured or forced between the glass plates with a?

A

pipet or syringe

72
Q

This is then placed on the top of the gel

A

comb

73
Q

For light-activated polymerization, the gel between the glass plates is exposed to a?

A

light source

74
Q

During light-activated polymerization, it is important to prevent air from getting into the gel or beneath the comb, why?

A

Bubbles will form discontinuities in the gel and oxygen will inhibit the polymerization of the acrylamide

75
Q

The comb is of a thickness equal to that of the _____ so that the gel will be the same thickness throughout.

A

spacers

76
Q

____________ is usually polyacrylamide while ___________ could either be agarose or polyacrylamide gels

A

Vertical gel; horizontal gel

77
Q

Used to monitor the progress of the electrophoresis run

A

Tracking dye

78
Q

The dyes migrate at specific speeds in a given gel concentration and usually run ahead of the smallest fragments of DNA

A

Tracking dye

79
Q

Examples of density agents

A

ficoll, sucrose or glycerol

80
Q

Gel loading is composed of

A
  1. Tracking Dye
  2. Density agent
81
Q

Increases the density of the sample as compared with the electrophoresis buffer

A

Density agent

82
Q

Two Most Common Tracking Dye

A
  1. Bromophenol blue
  2. Xylene cyanol green
83
Q

A tracking dye that is used for many applications

A

Bromophenol blue

84
Q

Another of the chromophores used as tracking dye for both agarose and polyacrylamide gels

A

Xylene cyanol green

85
Q

Electrophoresis is terminated when?

A

When tracking dye approaches the end of the gel or the desired distance

86
Q

Agents used most frequently for visualization of bands after electrophoresis

A
  • Fluorescent dyes
  • Silver stain

Both are associated with nucleic acid

87
Q

Most widely used dye in early DNA and RNA analyses

A

Ethidium Bromide

88
Q

EtBr is excited by UV light at ______ and in DNA emits visible light at ______

A

300 nm; 590 nm

89
Q

DNA separated in agarose or acrylamide and exposed to EtBr will emit ____ light when illuminated at _____

A

orange; 300 nm

90
Q

After electrophoresis, the agarose or acrylamide gel is soaked in a solution of _________ in running buffer (TAE, TBE, or TPE) or in TE.

A

0.1- to 1 mg/mL EtBr

91
Q

Q1: Used for dsDNA

Q2: Used for ssDNA, RNA

A

Q1: SYBR Green I

Q2: SYBR Green II

92
Q

This differs from EtBr in that it does not intercalate between bases; it sits in the minor groove of the double helix

A

SYBR Green I

93
Q

SYBR green in association with DNA or RNA also emits light in the _____ range at ______nm.

A

orange; 522 nm

94
Q

In agarose gel electrophoresis, SYBR staining is _______ times more sensitive than EtBr

A

25 to 100 times

95
Q

This can also be added directly to the DNA sample before electrophoresis

A

SYBR green

96
Q

This decreases the amount of dye required for DNA visualization but lowers the sensitivity of detection and may, at higher DNA concentrations, interfere with DNA migration through the gel

A

DNA prestaining (SYBR green)

97
Q

Good for real time PCR

A

SYBR Green

98
Q

Because ______ is not an intercalating agent, it is not as mutagenic and is therefore safer to use.

A

SYBR green

98
Q

In SYBR Gold, fluorescence increase more than _____ - fold upon binding to double or single-stranded DNA or to RNA

A

1000- fold

99
Q

Types of silver stains

A
  1. Silver diamine
  2. Silver nitrate
99
Q

Like SYBR green, SYBR gold is excited by UV light at ______ wavelength

A

300 nm

100
Q

A sensitive staining system originally developed for protein visualization

A

Silver Stain

101
Q

When using silver stain, after electrophoresis, the sample is fixed with?

A

Methanol and acetic acid

102
Q

When using silver stain, after the sample has been fixed, the gel is then impregnated with _______ in a weakly acid solution

A

ammoniacal silver (silver diamine) solutions or silver nitrate

103
Q

Interaction of silver ions with acidic or nucleophilic groups on the target results in ____________, under optimal pH conditions

A

crystallization or deposition of metallic silver

104
Q

This precipitates upon introduction of formaldehyde in a weak acid solution, or alkaline solution for silver nitrate

A

Insoluble black silver salt

105
Q

Best used for thick gels

A

Silver diamine

106
Q

Considered to be more stable (A silver stain)

A

Silver nitrate

107
Q

These are especially useful for protein analysis and for detection of limiting amounts of product/ amplicons

A

Silver stains (silver diamine & silver nitrate)

108
Q

SYBR Gold emits light at?

A

537 nm

SECOND SEM 3rd YEAR (38 decks)

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