Lesson 4 - Gel Electrophoresis

Question 1

• movement of electrically charged molecules in an electric field
• allows molecular separation based on size and net charge
• most common method for separation and visualization of DNA

Answer 1

gel electrophoresis

Question 2

gel electrophoresis allows molecular separation based on

Answer 2

1. size
2. net charge

Question 3

anion -> __

Answer 3

anode

Question 4

cation -> __

Answer 4

cathode

Question 5

what is used in gel electrophoresis

Answer 5

gel (molecular sieve)

Question 6

example of gels used in electrophoresis

Answer 6

1. agarose
2. acrylamide

Question 7

agarose are used for what?

Answer 7

large fragments of DNA

Question 8

acrylamide are used for what?

Answer 8

shorter fragments (1-1000 base pairs)

Question 9

used to put holes in gel

Answer 9

comb

Question 10

what is the gel stained with

Answer 10

colored or fluorescent dye

Question 11

where is the gel exposed to after dyeing

Answer 11

UV light

Question 12

DNA fluoresces as __ __

Answer 12

red orange

Question 13

rate of DNA when it travels to the anode (+)

Answer 13

inversely proportional to its molecular weight

Question 14

intercalates DNA due to its planar structure

Answer 14

ethidium bromide (EtBr)

Question 15

EtBr at UV lights emits a red-orange color at __ nm

Answer 15

590 nm

Question 16

buffers for electrophoresis

Answer 16

1. Tris-Acetate EDTA
2. Tris-Borate EDTA

Question 17

• common running buffer for agarose
• working solution: 0.5-1x
• stock solution: 50 x

Answer 17

Tris-Acetate EDTA

Question 18

Tris-Acetate EDTA is a common running buffer for what type of gel

Answer 18

agarose

Question 19

working solution of Tris-Acetate EDTA

Answer 19

0.5 - 1x

Question 20

stock solution of Tris-Acetate EDTA

Answer 20

50x

Question 21

• stronger buffering capacity than TAE
• can be used for agarose or PAGE
• stock solution: 5

Answer 21

Tris-Borate EDTA

Question 22

where can Tris-Borate EDTA be used

Answer 22

• Agarose
• polyacrylamide gels

Question 23

stock solution of Tris-Borate EDTA

Answer 23

5x

Question 24

stain placed in isolate

Answer 24

blue juice (bromophenol blue)

Question 25

amount of stain placed in isolate

Answer 25

1-2 μL

Question 26

agarose gel prep

Answer 26

agarose powder + buffer

Question 27

where is the agarose solution cooked in

Answer 27

microwave

Question 28

used in molecular biology labs to view DNA (or RNA) that has been separated by electrophoresis through an agarose gel

Answer 28

UV-transilluminators

Question 29

How is the DNA thought to travel through the gel pores

Answer 29

end-on fashion

Question 30

end-on movement of DNA is sometimes referred to as:

Answer 30

- snaking - reptation

Question 31

are more likely to be trapped in the gel

Answer 31

larger DNA molecules

Question 32

size of DNA molecules that run in about the same place in a conventional agarose gel

Answer 32

30 kbp (kilo base pair)

Question 33

Three types of DNA topology

Answer 33

1. Supercoiled DNA 2. nicked DNA 3. linear DNA

Question 34

highly compacted and runs rapidly through the gel

Answer 34

supercoiled DNA

Question 35

- molecule adopts an open structure - low mobility

Answer 35

nicked DNA

Question 36

what is a nick in DNA

Answer 36

single break in one strand of sugar-phosphate backbone

Question 37

- DNA backbone is broken in both strands - runs with intermediate mobility

Answer 37

linear DNA

Question 38

how is plasmid isolated

Answer 38

1. SDS + sodium hydroxide 2. neutralize with potassium acetate 3. remove precipitate by centrifugation 4. plasmid DNA in solution

Question 39

Other gel stains that are not as carcinogenic as ethidium bromide

Answer 39

- gel red - SYBR safe (1% or 1.5%)