Lecture 1 - Gel Electrophoresis

Question 1

What is the purpose of subjecting DNA molecules to agarose gel-electrophoresis?

Answer 1

To separate DNA molecules based on their molecular mass or size in base pairs.

Question 2

What around what pH is DNA negatively charged?

Answer 2

around neutral pH (~pH 7)

Question 3

As DNA is negatively charged, what electrode will the DNA move towards in an electrode field?

Answer 3

The positive electrode

Question 4

What is agarose?

Answer 4

an uncharged polysaccharide purified from agar of the seaweed, Agar agar

Question 5

What is the structure of an agarose gel matrix?

Answer 5

• if you heat molecules up, they go into solutions and start to form helical chains
• the helical chains start to come together to form a matrix
• the matrix is used to separate DNA molecules

Question 6

What are the steps for preparing an agarose gel?

Answer 6

1) The gel tray
2) prepare barriers to retain the agarose
3) pour molten agarose into the tray
4) insert comb to form the wells before the agarose solidifies
5) load DNA samples in individual wells and apply voltage

Question 7

Which size DNA molecules migrate more rapidly? Why?

Answer 7

• small sized DNA molecules migrate more rapidly than larger sized DNA molecules
• the pores in the agarose gel-matrix impede the larger molecules form migrating as far as the smaller molecules

Question 8

What can be used to extrapolate the molecular mass (# of base-pairs) of an unknown DNA fragment?

Answer 8

The generation of a standard curve of known molecular mass (# of base-pairs). It’s the first stage in the characterization of an unknown DNA molecule.

Question 9

What are the parameters that influence the rate of migration of DNA molecules during gel-electrophoresis?

Answer 9

1) percentage of agarose in the gel matrix
2) topology (conformation) of the DNA molecule; linear, relaxed circular, or super coiled
3) the voltage applied (

Question 10

What does the higher the concentration of agarose correlate with?

Answer 10

The smaller the average pore size in the gel-matrix

Question 11

What does the pore size of the gel-matrix affect?

Answer 11

the effective separation range of linear DNA molecules during gel-electrophoresis

Question 12

What does making smaller pores in the gel-matrix do?

Answer 12

changes the actual separation range

Question 13

What does the topology (conformation) of the DNA molecule mean?

Answer 13

the shape of the DNA molecule. there’s 3 shapes. linear, relaxed circular and supercoiled

Question 14

What are characteristics of supercoiled DNA molecules?

Answer 14

• can either be circular or linear, but the ends of the linear molecule must be restrained
• in cells, DNA is often negatively supercoiled
• positively supercoiled DNA can be produced in vitro

Question 15

What are the parameters that DO NOT influence the rate of migration of DNA molecules during agarose gel-electrophoresis?

Answer 15

• the %GC or sequence of a DNA molecule

Question 16

Why map recognition sites for restriction endonucleases?

Answer 16

it is often the first step in the characterization of an unknown DNA molecule, leading to further manipulatinos, e.g., cloning and DNA sequencing