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BIO2030 Biotechnology > Visualising DNA > Flashcards

Flashcards in Visualising DNA Deck (9)
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1

What does gel electrophoresis do?

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

2

How does gel electrophoresis work?

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

3

How do you get a higher resolution on gel electrophoresis?

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

4

What is pulsed-field gel electrophoresis?

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

5

Name 3 ways DNA can be denatured?

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

6

What is hybridisation?

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

7

What are the conditions favouring base pairing in hybridisation?

Low temperature
Higher [salt]
Removal of denaturants

8

What is Southern blotting?

A way of analysing mRNA

9

Describe Southern blotting's mechanism

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