Gel electrophoresis and blotting

What does TRIS do?

It is a pH buffer (holds pH at somewhere between 7-9)

What can happen to DNA if the pH is too high?

Separates into single strands

What can happen to DNA if the pH is too low?

Creates abasic sites

When is urea used in gel electrophoresis?

When you want the DNA in its single stranded form

What does EDTA do in gel electrophoresis?

Forms a molecular cage around divalent cations (such as those in DNAses) which prevents DNA from being broken down

What does EDTA stand for?

Ethylene diamine tetraacetic acid

What do sodium borate or sodium acetate do in the gel?

Allow for conduction of electricity

What are the two main chemicals used to make gels?

• Agarose from seaweed (safe but low res)

• Acrylamide and bis acrylamide (high resolution but neurotoxic)

How is agarose set?

By cooling

How is acrylamide set?

With chemicals

What is the danger of acrylamide?

It can polymerise in human tissue and rupture myelin sheaths of cells- can cause paralysis

What does ethidium bromide do in a gel electrophoresis experiment?

Fluoresces more when stacked in between DNA bases than when it’s on its own and makes the bands visible 

At what wavelength does ethidium bromide fluoresce?

590nm

What is a transilluminator?

A box with a quartz window which allows UV light through

What is the relationship between log(size of DNA) and how far the DNA has moved?

Inverse linear relationship

What are the three states of plasmid DNA

Supercoiled

Nicked (relaxed) circular

Linear

What is nucleic acid hybridisation?

The tendency for single strands of DNA or RNA to hybridise with complementary sequences

What is Southern blotting used for?

Finding a particular sequence when you have a mix of sequences that have been run through a gel. This used to be the basis of genome sequencing, comparison of DNA between species, forensics and DNA profiling. 

What are Northern and Western blotting?

Like Southern blotting but for RNA and proteins respectively.

Describe how Southern blotting works

Detection is done by hybridisation using a radioactive or fluorescent probe

A DNA probe is hybridised to a DNA sample

Why is an alkaline solution used in Southern blotting?

To separate DNA strands

What is nitrocellulose used for in Southern blotting?

DNA sticks to it

Give a use of Southern blotting in diagnostics

Beta thalassaemia and sickle cell disease are both mutations in the beta globin gene- if someone is carrying a mutation for either they will have different sized DNA fragments than non-carriers

What are the applications of Northern blotting

Shows which genes are being transcribed in which tissues at a particular time

What is an expression array?

A visual comparison of which genes are being transcribed in one, the other, or both of two sets of tissues