Wright 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

DNA at neutral pH

Answer 2

Negatively charged because of phosphate groups

Will move towards positive electrode in an electric field

Question 3

Agarose

Answer 3

Uncharged polysaccharide purified from agar of seaweed Agar gar
Form helical chains at high temperature and forms matrix with pores as it cools

Question 4

Preparation of agarose gel

Answer 4

1. Gel tray
2. Prepare barriers to retain agarose in tray
3. Insert comb to form wells before agarose solidifies
4. Load DNA samples into individual wells and apply voltage

Question 5

Ethidium Bromide

Answer 5

Binds to DNA to visualize using UV light

Mutagen

Question 6

Linear DNA migration

Answer 6

Migrate at rate that is inversely proportional to the log10 of their molecular mass or base pairs
Smaller sized molecules migrate more rapidly

Question 7

Standard curve

Answer 7

Can generate curve of known molecular mass of DNA and migration to extrapolate size of unknown DNA fragment
Usually first stage of characterization of unknown DNA molecule

Question 8

Parameters influencing rate of DNA migration

Answer 8

1. Percentage of agarose
2. Topology
3. Voltage applied

Question 9

Percentage of agarose in gel matrix

Answer 9

Higher concentrations of agarose, smaller average pore size in gel matrix
Pore size of gel matrix affects the effective separation range of linear DNA molecules during gel electrophoresis

Question 10

Topology

Answer 10

Conformation of DNA molecule

Linear, relaxed circular, supercoiled

Question 11

Topoisomerase

Answer 11

Induces supercoiling
Circular or linear DNA
Ends of linear molecules must be restrained

Question 12

Negative supercoiled DNA

Answer 12

Main form in cells

Question 13

Positively supercoiled DNA

Answer 13

Produced in vitro

Question 14

Voltage applied

Answer 14

Distortion of rate of movement of higher molecular weight molecules
Will not have linear arrangement
Can melt gel

Question 15

Parameters that do not influence rate of migration of DNA molecules

Answer 15

%GC content

Question 16

Restriction endonucleases

Answer 16

Enzymes that cleave (cut) DNA at specific sequences
Found naturally in bacteria and some yeasts to prevent infection from DNA viruses
Recognize palindromic sequences

Question 17

Sticky ends

Answer 17

Overhands of DNA

5’ or 3’

Question 18

Blunt ends

Answer 18

No sticky ends of DNA

Question 19

Restriction endonuclease nomenclature

Answer 19

Based on organism of origin, strain, and number of enzymes isolated from that organism

Question 20

Restriction map sites

Answer 20

Often first step in characterizing unknown DNA molecule to be further manipulated