Visualising DNA

Question 1

What does gel electrophoresis do?

Answer 1

Separates DNA fragments of different sizes
Needs a chemical dye to see the DNA;
Usually ethidium bromide or SYBR green or gold

Question 2

How does gel electrophoresis work?

Answer 2

DNA moves through the gel at a rate that is inversely proportional to the chain length of the DNA

Question 3

How do you get a higher resolution on gel electrophoresis?

Answer 3

Using radioactive labelled DNA run on a polyacrylamide gel followed by autoradiography

Question 4

What is pulsed-field gel electrophoresis?

Answer 4

Separation of large DNA molecules by periodically changing the electric field from a gel matrix
Alternating voltage improves the resolution of larger molecules
The voltage is switched between 3 directions;
The central axis of the gel and 60 degrees on each side of the central axis
The electric field alternates 120 degrees every 90 seconds for 18-24 hours at 14 degrees Celcius
Thus the DNA doesn’t move in a straight line but in a net forward migration pattern

Question 5

Name 3 ways DNA can be denatured?

Answer 5

Heat- DNA with higher GC content denatures at a higher temperature
Extreme pH
Conc. urea

Question 6

What is hybridisation?

Answer 6

The process by which separated DNA/RNA will reform a double helix

Question 7

What are the conditions favouring base pairing in hybridisation?

Answer 7

Low temperature
Higher [salt]
Removal of denaturants

Question 8

What is Southern blotting?

Answer 8

A way of analysing mRNA

Question 9

Describe Southern blotting’s mechanism

Answer 9

RNA is separated by agarose gel electrophoresis
RNA is blotted onto nylon or nitrocellulose membrane
Incubate membrane with labelled probe
Locate hybridising bands by autoradiography