AGAROSE GEL ELECTROPHORESIS

Question 1

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

Answer 1

ELECTROPHORESIS

Question 2

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

Answer 2

the molecular size and net electrical charge

Question 3

attraction or repulsion between charged particles

Answer 3

Electrostatic Force

Question 4

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

Answer 4

Friction Force

Question 5

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

Answer 5

Electrophoretic Retardation Force

Question 6

Electrostatic Force (Fe) equation

Answer 6

𝐹𝑒 = 𝐸 × 𝑞
E = electric field (V/m)
q = charge of particles (C)

Question 7

Drag Force (Fd) equation

Answer 7

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

Question 8

Electrophoretic
Mobility (μ)

Answer 8

𝜇 = 𝑞/𝑓 = 𝑣/𝐸

Question 9

Velocity
of the
particle

Answer 9

𝑣 = (𝑞 × 𝐸)/𝑓

Question 10

Charges will migrate
to their opposite
pole

Answer 10

Net Charge

Question 11

Smaller molecules
will travel farther

Answer 11

Molecule
size

Question 12

Denatured DNA will
migrate more
predictably according
to size

Answer 12

Molecule
Shape

Question 13

𝑉 = 𝐼𝑅

Answer 13

Applied
Voltage

Question 14

composition and
properties of the
buffer affects
electrophoretic
mobility

Answer 14

The buffer

Question 15

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

Answer 15

The Gel

Question 16

Sulfonated polysaccharide polymer
from seaweed

Answer 16

Agarose Gel

Question 17

Agarose Gel

• Linear polymer of ________
  consisting:
• ___________
• ___________

Answer 17

agarobiose

• 1,3-linked-β-D-galactopyranose
• 1,4-linked-3,6-anhydro-α-Lgalactopyranose

Question 18

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 = _____

Answer 18

1. 55-65 oC
2. Comb
3. ~40 oC
4. smaller pores

Question 19

• 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)

Answer 19

Polyacrylamide

Question 20

• 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?

Answer 20

1. RUNNING Buffer
2. pH 8.0

Question 21

Henderson Hasselbach Equation

Answer 21

𝑝𝐻 = 𝑝𝐾𝑎 + 𝑙𝑜𝑔 ([𝐴−]/[𝐻𝐴])

Question 22

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

Answer 22

T

Question 23

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

Answer 23

F - greater

Question 24

Solution: Higher buffer concentrations ran at a higher voltage

Answer 24

F - lower

Question 25

* Most commonly used for DNA electrophoresis * DNA migrates faster in TAE * More stable when stored

Answer 25

TRIS Acetate EDTA (TAE)

Question 26

* Greater buffering capacity * Stock solutions are prone to precipitation

Answer 26

Tris borate EDTA (TBE)

Question 27

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

Answer 27

salt wave

Question 28

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 *____ *____

Answer 28

1. Denaturation 2. Formamide 3. Urea

Question 29

* Contain a colored dye and a density agent

Answer 29

Loading Buffer

Question 30

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

Answer 30

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

Question 31

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

Answer 31

Tracking dye

Question 32

* 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

Answer 32

1. gel boxes 2. Electrodes

Question 33

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

Answer 33

1. loading buffer 2. 25 μL

Question 34

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

Answer 34

Dyeing your gel

Question 35

* Intercalating agent * Used to be the most widely used dye * Highly carcinogenic

Answer 35

Ethidium bromide

Question 36

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

Answer 36

1. 590 nm 2. 300 nm

Question 37

* 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

Answer 37

SYBR Green

Question 38

SYBR Green double-stranded DNA (used in RT-PCR)

Answer 38

SyBr Green I

Question 39

SYBR Green Single-stranded DNA or RNA staining

Answer 39

SyBr Green II

Question 40

SYBR Green Can be used for both DNA and RNA staining

Answer 40

SyBr Gold

Question 41

SYBR Green Variant of SyBr Green that has been deemed as nonhazardous waste

Answer 41

SyBr Safe

Question 42

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

Answer 42

Gel Red

Question 43

* Similar absorption and emission spectra as EtBr * Can be added to the gel mix like SyBr Green * Cheaper than SyBr Green

Answer 43

Gel Red (gihapon)

Question 44

* 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

Answer 44

Silver Stain