Quiz 2: Lab Agarose Gel Electrophoresis

Question 1

What is Agarose gel electrophoresis the most effective way of?

Answer 1

Separating DNA fragments of varying sizes ranging from 100 bp to 25 kb

Question 2

What happens to agarose polymers during gelation?

Answer 2

During gelation, agarose polymers associate non-covalently and form a network of bundles whose pore sizes determine a gel’s molecular sieving properties

Question 3

What did the use of agarose gel revolutionize?

Answer 3

Separation of DNA

Question 4

What is done to separate DNA using agarose gel electrophoresis?

Answer 4

DNA is loaded into pre-cast wells in the gel and a current applied
The phosphate backbone of the DNA (and RNA) molecule is negatively charged, therefore when placed in an electric field, DNA fragments will migrate to the positively charged anode

Because DNA has a uniform mass/charge ratio, DNA molecules are separated by size within an agarose gel in a pattern such that the distance traveled is inversely proportional to the log of its molecular weight

Question 5

What is the leading model for DNA movement through agarose gel?

Answer 5

“Biased Reptation” whereby the leading edge moves forward and pulls the rest of the molecule along

Question 6

The rate of migration of a DNA molecule through a gel is determined by the following: (7)

Answer 6

1) size of DNA molecule
2) agarose concentration
3) DNA conformation
4) voltage applied
5) presence of ethidium bromide
6) type of agarose
7) electrophoresis buffer

Question 7

How can the DNA molecules be visualized after separation?

Answer 7

Under UV light after staining with an appropriate dye

Question 8

By following this protocol, students should be able to: (8)

Answer 8

1. Understand the mechanism by which DNA fragments are separated within a gel matrix
2. Understand how conformation of the DNA molecule will determine its mobility through a gel matrix
3. Identify an agarose solution of appropriate concentration for their needs
4. Prepare an agarose gel for electrophoresis of DNA samples
5. Set up the gel electrophoresis apparatus and power supply
6. Select an appropriate voltage for the separation of DNA fragments
7. Understand the mechanism by which ethidium bromide allows for the visualization of DNA bands
8. Determine the sizes of separated DNA fragments

Question 9

What does loading dye help to do?

Answer 9

Track how far your DNA sample has traveled, and allows the sample to sink into the gel

Question 10

What do you do with the running buffer?

Answer 10

Add enough running buffer to cover the surface of the gel
Important to use the same running buffer as the one used to prepare the gel

Question 11

What should always be loaded along with the experimental samples?

Answer 11

Appropriate DNA size marker (DNA standard/ DNA Ladder)

Question 12

What are the cathode and anode?

Answer 12

Cathode (Black leads) should be closer to the wells than the anode
Anode (Red leads)

Question 13

Agarose gel electrophoresis has proven to be an efficient and effective way of what?

Answer 13

Separating nucleic acids

Question 14

What is molecular sieving determined by?

Answer 14

Size of pores generated by the bundles of agarose in the gel matrix

Question 15

The higher the concentration of agarose,

Answer 15

The smaller the pore size

Question 16

What is EtBr?

Answer 16

Most common reagent used to stain DNA in agarose gels

Question 17

How does EtBr work?
What does it allow for?

Answer 17

EtBr works by intercalating itself in the DNA molecule in a concentration dependent manner
This allows for an estimation of the amount of DNA in any particular DNA band based on its intensity
(Because of its positive charge, the use of EtBr is reduces DNA migration by 15%)

Question 18

What stain did we use for DNA in agarose gel?

Answer 18

Methyl blue?

Question 19

What are the 3 major purposes of loading dyes used in gel electrophoresis?

Answer 19

Add density to the sample, allowing it to sink into the gel
Dyes provide color and simplify the loading process
Dyes move at standard rates through the gel, allowing for the estimation of the distance that DNA fragments have migrated

Question 20

How can the exact sizes of separated DNA fragments be determined?

Answer 20

By plotting the lot of the molecular weight for the different bands of a DNA standard against the distance traveled by each band

Question 21

What does the DNA standard (ladder) contain?

Answer 21

Mixture of DNA fragments of pre-determined sizes that can be compared against the unknown DNA samples

Question 22

What is important to note?

What moves through the gel the fastest?

What moves the slowest?

Answer 22

Different forms of DNA move through the gel at different rates

Supercooled plasmid DNA, because of its compact formation
Followed by a linear DNA fragment of the same size

Open circular form traveling the slowest

Question 23

What are the most common gel running buffers?

Answer 23

TAE (40 mM Tris-acetate, 1 mM EDTA) and TBE (45 mM Tris-borate, 1 mM EDTA)