AGE

Question 1

Electrophoresis

Movement of dispersed, charged particles relative to a fluid under the influence of a spatially uniform____

Answer 1

electric field

Question 2

Used in the separation of (3) based on the _____and _____

Answer 2

DNA, RNA, or proteins

molecular size and net electrical charge

Question 3

• electric field,
• migration

Most biomolecules exist as electrically-charged particles

Answer 3

ELECTRO

PHORESIS

Question 4

• attraction or repulsion between charged particles

Answer 4

Electrostatic Force

Question 5

Electrostatic force =

Answer 5

Coulomb force

Question 6

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

Answer 6

Friction Force

Question 7

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

Answer 7

Electrophoretic Retardation Force

Question 8

is a force that slows down the movement of charged particles in a solution.

It occurs when charged particles move in one direction, while the ions around them move in opposite direction

Answer 8

Electrophoretic retardation force

Question 9

• solute response to applied electric field

Answer 9

Electrophoretic mobility

Question 10

_____acting on an object moving in a fluid oppose the motion. Ex.
Aerodynamic force that opposes an aircraft’s motion through the air.

Answer 10

Drag forces

Question 11

ELECTROSTATIC FORCE (Fe)

DRAG FORCE (Fd)

Answer 11

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

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

Question 12

NET CHARGE
Charges will migrate to their…

Answer 12

opposite pole

Question 13

MOLECULAR SIZE
Smaller molecules will…

Answer 13

travel farther

Question 14

MOLECULE SHAPE
Denatured DNA will migrate…

Answer 14

more predictably according to size

Question 15

APPLIED VOLTAGE

Formula

Answer 15

V= IR

Question 16

Composition and properties of the ___ affects electrophoretic mobility

Answer 16

THE BUFFER

Question 17

THE GEL
______ of the gel will determine the ease of movement of differently sized macromolecules

Answer 17

Pore size

Question 18

FACTORS AFFECTING A.G.E.

Answer 18

NET CHARGE
MOLECULAR SIZE MOLECULE SHAPE
APPLIED VOLTAGE
THE BUFFER
THE GEL

Question 19

AGAROSE GEL

Sulfonated polysaccharide polymer from…

Answer 19

seaweed

Question 20

AGAROSE GEL

Linear polymer of_____ consisting:
(2)
It is a disaccharide (combination of two monosaccharides).

Answer 20

agarobiose

1,3-linked-ß-D-galactopyranose
1,4-linked-3,6-anhydro-a-L-galactopyranose

Question 21

______ is made up of a repeating unit of D-galactose and 3, 6-anhydro-L-galactopyranose joined by 1-4 linkages.

Answer 21

Agarobiose

Question 22

Agarobiose is made up of a repeating unit of______ and _____ joined by_____

Answer 22

D-galactose and 3, 6-anhydro-L-galactopyranose

1-4 linkages.

Question 23

Agarose

Process:
Dried powder is dissolved in hot running____
Cooled between____ then poured to a casting tray
_____is inserted to create wells for the sample
_____(comb appearance)
Solidifies at____

Answer 23

buffer

55-650C

Comb

Comb-molder

~40C

Question 24

_______ dictates the size of the spaces in the gel (pore size)

Higher concentration =_____

Answer 24

Concentration

smaller pores

Question 25

Separation of linear, double-stranded DNA molecules in TAE agarose with regular voltage below___

Answer 25

5 V/cm.

Question 26

- Better for proteins - Components are synthetic, causing less differences in batches from different sources - Higher resolution of smaller fragments

Answer 26

Polyacrylamide

Question 27

- Powdered form is a potent neurotoxin - Difficult gel preparation (thinner gels)

Answer 27

Polyacrylamide

Question 28

- organic chemicals: pharmaceuticals and carbohydrates, inorganic anions and metals

Answer 28

Capillary

Question 29

- Increased sensitivity - Immediate detection - multiple color detection systems - Decreased labor and run time

Answer 29

Capillary

Question 30

Instruments used are very costly compared to AGE

Answer 30

Capillary

Question 31

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 (pH 8.0 for nucleic acids)

Answer 31

Running buffer

Question 32

In order to change the pH of a buffered solution by 1 point, the acidic or basic form of the buffer must be brought to a concentration 1/10 that of the other form - basically like diluting to 1/10 to change the pH

Answer 32

Henderson-Hasselbach Equation

Question 33

^ Buffer concentration =_____ Increase in concentration of the buffer causes increased_____ and offers greater____ stability Greater conductivity results in greater heat generation at a given voltage Solution:…

Answer 33

^ HEAT conductivity; pH Higher buffer concentrations ran at a lower voltage

Question 34

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

Answer 34

TRIS Acetate EDTA (TAE)

Question 35

Greater buffering capacity Stock solutions are prone to ***precipitation*** Causes a ***gradient concentration difference*** that distorts migration patterns (salt wave)

Answer 35

Tris borate EDTA (TBE)

Question 36

-Denaturation 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 36

BUFFER ADDITIVES

Question 37

BUFFER ADDITIVES

Answer 37

• Formamide • Urea

Question 38

• Results in long, straight, unpaired chains that have a more predictable migration through the gel

Answer 38

Buffer additives

Question 39

(e.g. Ficoll, sucrose, or glycerol) to ***increase the density of the sample*** in comparison to the buffer ***monitors the progress of migration*** (e.g. Bromophenol blue)

Answer 39

- Density agents -Tracking dye

Question 40

(e.g. Ficoll, sucrose, or glycerol) to ***increase the density of the sample*** in comparison to the buffer ***monitors the progress of migration*** (e.g. Bromophenol blue)

Answer 40

- Density agents -Tracking dye

Question 41

EQUIPMENT ______are ran horizontally in acrylic containers (gel boxes) ______in the gel compartment are connected to a power supply The gel is placed in the middle and submerged in the_____, filing both compartments

Answer 41

Agarose gels Electrodes running buffer

Question 42

GEL LOADING LOADING THE SAMPLES ✔ Dilute the samples in your loading buffer 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 ✔ Make sure your tip is within the well before loading ✔ When withdrawing the tip, try to go straight up ✔ Take care to not pierce the gel all the way down

Answer 42

25 μL

Question 43

DETECTION SYSTEMS DYEING YOUR GEL After electrophoresis, the bands are visualized using dyes that specifically associate with nucleic acids (2)

Answer 43

fluorescent dyes and silver stain

Question 44

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

Answer 44

ETHIDIUM BROMIDE

Question 45

Ethidium Bromide - Emits visible light at ______when excited at UV light of____

Answer 45

590 nm (orange) 300 nm

Question 46

- 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 46

SYBR GREEN

Question 47

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

Answer 47

- SyBr Safe

Question 48

• Can be used for both DNA and RNA staining

Answer 48

- SyBr Gold

Question 49

• Single-stranded DNA or RNA staining

Answer 49

- SyBr Green Il

Question 50

• double-stranded DNA (used in rt-PCR)

Answer 50

- SyBr Green I

Question 51

- Intercalating agent - ***Cannot penetrate the cell membrane*** - ***Higher sensitivity than EtBr***

Answer 51

GEL RED

Question 52

- Can be used for ***dsDNA, ssDNA, or RNA*** - Similar absorption and emission spectra as EtBr - Can be added to the gel mix like SyBr Green ***Cheaper than SyBr Green*** • 136 USD = 7.6k (for 0.5 mL before taxes and shipping) • SyBr Green 155 USD = 8.7k (for 0.5 mL before taxes and shipping)

Answer 52

Gel Red

Question 53

- ***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 53

SILVER STAIN

Question 54

_______is usually 10-100 times more sensitive than Coomassie Blue staining, but it is more complicated. Faint but still visible bands on this gel contain less than 0.5 ng of protein!

Answer 54

Silver staining