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MOLBIO LEC MIDTERMS > AGAROSE GEL ELECTROPHORESIS > Flashcards

AGAROSE GEL ELECTROPHORESIS Flashcards

1
Q

Movement of dispersed, charged particles relative to a fluid under the influence of a spatially uniform electric field

A

ELECTROPHORESIS

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2
Q

Used in the separation of DNA, RNA, or proteins based on – and –

A

the molecular size and net electrical charge

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3
Q

attraction or repulsion between charged particles

A

Electrostatic Force

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4
Q

Resistance to motion when the surface of one object comes in contact with another

A

Friction Force

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5
Q

Force exerted by the medium on particles in a direction opposite to particle movement

A

Electrophoretic Retardation Force

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6
Q

Electrostatic Force (Fe) equation

A

𝐹𝑒 = 𝐸 Γ— π‘ž
E = electric field (V/m)
q = charge of particles (C)

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7
Q

Drag Force (Fd) equation

A

𝐹𝑑 = 𝑓 Γ— 𝑣
f = frictional coefficient
v = velocity of the particle (m/s)

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8
Q

Electrophoretic
Mobility (ΞΌ)

A

πœ‡ = π‘ž/𝑓 = 𝑣/𝐸

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9
Q

Velocity
of the
particle

A

𝑣 = (π‘ž Γ— 𝐸)/𝑓

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10
Q

Charges will migrate
to their opposite
pole

A

Net Charge

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11
Q

Smaller molecules
will travel farther

A

Molecule
size

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12
Q

Denatured DNA will
migrate more
predictably according
to size

A

Molecule
Shape

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13
Q

𝑉 = 𝐼𝑅

A

Applied
Voltage

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14
Q

composition and
properties of the
buffer affects
electrophoretic
mobility

A

The buffer

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15
Q

Pore size of the gel
will determine the
ease of movement of
differently sized
macromolecules

A

The Gel

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16
Q

Sulfonated polysaccharide polymer
from seaweed

A

Agarose Gel

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17
Q

Agarose Gel

  • Linear polymer of ________
    consisting:
  • ___________
  • ___________
A

agarobiose

  • 1,3-linked-Ξ²-D-galactopyranose
  • 1,4-linked-3,6-anhydro-Ξ±-Lgalactopyranose
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18
Q

Agarose Gel

  • Process:
  • Dried powder is dissolved in hot running buffer
  • Cooled between ____ then poured to a casting tray
  • ____ is inserted to create wells for the sample
  • Solidifies at ____
  • Concentration dictates the size of the spaces in the gel (pore size)
  • Higher concentration = _____
A
  1. 55-65 oC
  2. Comb
  3. ~40 oC
  4. smaller pores
19
Q
  • Better for proteins
  • Components are synthetic, causing less differences in batches from different sources
    *Powdered form is a potent neurotoxin
    *Difficult gel preparation (thinner gels)
A

Polyacrylamide

20
Q
  • Carries the current and
    protects the samples
  • Weak acid and its conjugate
    base able to take up or release
    protons to maintain a constant
    pH (______ for nucleic acids)

what am I and what ph for nucleic acids?

A
  1. RUNNING Buffer
  2. pH 8.0
21
Q

Henderson Hasselbach Equation

A

𝑝𝐻 = π‘πΎπ‘Ž + π‘™π‘œπ‘” ([π΄βˆ’]/[𝐻𝐴])

22
Q

T or F:
Increase in concentration of the buffer causes increased conductivity and offers greater pH stability

A

T

23
Q

T or F:
Greater conductivity results in lesser heat generation at a given voltage

A

F - greater

24
Q

Solution: Higher buffer concentrations ran at a higher voltage

A

F - lower

25
Q
  • Most commonly used for DNA electrophoresis
  • DNA migrates faster in TAE
  • More stable when stored
A

TRIS Acetate EDTA
(TAE)

26
Q
  • Greater buffering capacity
  • Stock solutions are prone to
    precipitation
A

Tris borate EDTA
(TBE)

27
Q

Tris borate EDTA (TBE)
* Causes a gradient
concentration difference
that distorts migration
patterns (_________)

A

salt wave

28
Q

Buffer additives

*________agents are commonly used for nucleic acids to break down H-bonds between complementary strands or within the same strand of DNA or RNA
*____
*____

A
  1. Denaturation
  2. Formamide
  3. Urea
29
Q
  • Contain a colored dye and a density agent
A

Loading Buffer

30
Q

_____ to increase the density of the
sample in comparison to the buffer

A

Density agents (e.g. Ficoll, sucrose, or
glycerol)

31
Q

_____ monitors the progress of
migration (e.g. Bromophenol blue)

A

Tracking dye

32
Q
  • Agarose gels are ran horizontally in
    acrylic containers (_____)
  • ____ in the gel compartment are connected to a power supply
  • The gel is placed in the middle and
    submerged in the running buffer, filling
    both compartments
A
  1. gel boxes
  2. Electrodes
33
Q

Gel Loading- Loading the samples

βœ“ Dilute the samples in your ____ prior to loading
βœ“ Know the capacity of the well (usually, ___ is a safe volume for a >5 well format)
βœ“ Depress the plunger of the micropipette before loading and do not let go until you are done and out of the buffer

A
  1. loading buffer
  2. 25 ΞΌL
34
Q

After electrophoresis, the bands are visualized using dyes that specifically associate with nucleic acids (fluorescent dyes and silver stain)

A

Dyeing your gel

35
Q
  • Intercalating agent
  • Used to be the most widely used dye
  • Highly carcinogenic
A

Ethidium bromide

36
Q

Ethidium bromide

Emits visible light at ____
(orange) when excited at UV
light of ____

A
  1. 590 nm
  2. 300 nm
37
Q
  • Sits on the minor groove of the
    double helix
  • 25-100x more sensitive than
    EtBr
  • Can be added to the gel mix
  • Requires special optical filters
A

SYBR Green

38
Q

SYBR Green

double-stranded DNA (used in
RT-PCR)

A

SyBr Green I

39
Q

SYBR Green

Single-stranded DNA or RNA
staining

A

SyBr Green II

40
Q

SYBR Green

Can be used for both DNA and
RNA staining

A

SyBr Gold

41
Q

SYBR Green

Variant of SyBr Green that has
been deemed as nonhazardous waste

A

SyBr Safe

42
Q
  • Intercalating agent
  • Cannot penetrate the cell membrane
  • Higher sensitivity than EtBr
  • Can be used for dsDNA,
    ssDNA, or RNA
A

Gel Red

43
Q
  • Similar absorption and
    emission spectra as EtBr
  • Can be added to the gel mix
    like SyBr Green
  • Cheaper than SyBr Green
A

Gel Red (gihapon)

44
Q
  • Not an intercalating agent and
    is thus not toxic to humans
  • Considered a biohazard
  • More complicated than
    fluorescent stains
  • Increased sensitivity
  • Most useful in protein analysis
A

Silver Stain

MOLBIO LEC MIDTERMS (4 decks)

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