Electrophoresis

Question 1

Study of the movement of charged particles in an electric field

Answer 1

Electrophoresis

Question 2

Movement of a particle in an electric field is dependent on several factors including

Answer 2

Size, shape, charge and chemical makeup of the molecule

Question 3

Movement is represented by the equation

Answer 3

V= Eq/f

Question 4

V = Eq/f

Answer 4

V = velocity of the particle 
E = the electrical field in volts/cm
q = the charge on the particle (surface charge) in coulomb (c) or ampere-hour (Ah)
f = frictional coefficient (dependent on shape and mass of the particle)

Question 5

Electrophoresis is usually carried out under

Answer 5

constant voltage

Question 6

The movement of a charged particle depends only upon

Answer 6

the ratio q/f

Question 7

For a set of molecule of similar shape, f varies with

Answer 7

size but not shape

Question 8

A set of similar molecules will migrate in an electric field at a rate proportional to

Answer 8

their charge-to-mass ratio

Question 9

Carbohydrate extracted from seaweed

Answer 9

Agarose

Question 10

Gels of agarose as low as 0.5% can be prepared to yield a solid support capable of separating

Answer 10

Very large biomolecules

Question 11

The rapid advances in our understanding of nucleic acid structure and function is due in large part to

Answer 11

the development of gel electrophoresis

Question 12

Agarose gels are prepared by

Answer 12

Suspending dry agarose in aqueous buffers, heating until a clear solution is obtained, and allowing the solution to gel by cooling to room temperature

Question 13

The agarose gel has large pores, stabilized by

Answer 13

hydrogen bonds

Question 14

The pore size can be controlled by

Answer 14

the initial concentration of agarose

Question 15

Large pores are formed from

Answer 15

low concentrations

Question 16

Smaller pores are formed from

Answer 16

higher concentrations

Question 17

Provide a means to easily separate large molecular weight molecules such as DNA

Answer 17

Very large pore sizes of low concentration gels

Question 18

Tris-acetate (TAE) Buffer

Answer 18

(50X)
242gm trimethylamine (Tris) base
57.1 mL glacial acetic acid
100 mL 0.5M EDTA (pH 8.0) (Chelator)

Question 19

Chelator

Answer 19

0.5 M EDTA (pH 8.0)

Question 20

Moderately toxic and is a suspected carcinogen

Answer 20

Ethidium Bromide

Question 21

Intercalating ligand

Answer 21

Ethidium Bromide

Question 22

Ficoll Dye/Loading Dye

Answer 22

0.25% bromophenol blue
0.25% xylene cyanol
15% Ficoll dye (type 400) in water

Question 23

A water-soluble polysaccharide consisting of sucrose repeating units

Answer 23

Ficoll

Question 24

Ficoll dye purposes

Answer 24

1. Increases the density of the sample, ensuring that the DNA drops evenly into the well
2. Adds color to the sample, thereby simplifying the loading process
3. It contains dyes that move toward the anode at predictable rates

Question 25

Migrates through the agarose approximately 2.2-fold faster than xylene cyanol

Answer 25

Bromophenol blue

Question 26

Migrates at approximately the same rate as linear double-stranded 300bp DNA

Answer 26

Bromophenol blue

Question 27

Migrates at approximately the same rate as linear double-stranded 4kb DNA

Answer 27

Xylene cyanol FF

Question 28

3 to 60 kb Size of DNA fragments to be separated

Answer 28

0.3% Agarose

Question 29

1 to 30 kb Size of DNA fragments to be separated

Answer 29

0.5% Agarose

Question 30

0.8 to 12 kb Size of DNA fragments to be separated

Answer 30

0.7% Agarose

Question 31

0.5 to 10 kb Size of DNA fragments to be separated

Answer 31

1.0% Agarose

Question 32

0.4 to 3 kb Size of DNA fragments to be separated

Answer 32

1.2% Agarose

Question 33

0.2 to 3 kb Size of DNA fragments to be separated

Answer 33

1.5% Agarose

Question 34

For casting a 1 cm thick gel, combine the ________________ with agarose in the desired concentration to form a total volume of 50 mL

Answer 34

1X TAE Buffer

Question 35

Gel Casting and Chamber Prep

Answer 35

1. Pour Agarose solution into gel tray to the 1 cm mark 2. Allow gel to solidify and cool 3. Remove comb gently 4. Carefully remove rubber ends and position gel in the chamber with sample wells closest to the NEGATIVE electrode 5. Slowly fill chamber with 1X TAE buffer solution until gel is submerged (about 1mm)

Question 36

It is important to use the same batch of electrophoresis buffer in both

Answer 36

The electrophoresis chamber and the gel

Question 37

8 Place Comb

Answer 37

20 micro liters

Question 38

12 Place Comb

Answer 38

12 micro liters

Question 39

When using the 8 place comb, carefully add _______ of sample to the individual wells

Answer 39

10 micro liters

Question 40

Samples should contain

Answer 40

2 micro liters ficoll loading dye for visualization

Question 41

The leading dye should travel at least

Answer 41

two-thirds of the way through the gel

Question 42

Graph

Answer 42

``` X-axis = DNA migration (mm) y-axis = DNA base pairs or DNA log10 ```

Question 43

Picture taken of gel electrophoresis

Answer 43

Transillumination of DNA gel