Electrophoresis Flashcards
1
Q
How does electrophoresis work?
A
- DNA has negatively charged groups (PO4-)
- DNA will migrate towrds the poisitve electrode
- Mass/charge will be the same for all lengths of DNA
- length and size determines how fast the fragments travel
2
Q
How do we separate different lengths of DNA in gel electrophoresis?
A
- Pass them through a selective medium: a gel
- Fibrils hinder the movement of larger molecules
- Separates by size and shape since the mass/charge ratio is constant
- More compacted DNA will move faster than a rigid rod double helix
3
Q
More on electrophoretic separation
A
- Gels typically have a cathodic end, where the samples were loaded, at the top
- Using a ‘ladder’ lets you compare your sample with known lengths
- Migration distance can be calibrated to make accurate estimates of intermediate lengths
- Shorter strands move faster and appear at the bottom of the gel
4
Q
What types of gel are used in gel electrophoresis?
A
- Agarose = tangled fibrils (physical gel), heat-cool to cast, gel conc 0.5-2%, DNA size range 50-30,000 bp
- Poly(acrylamide) {PAGE} = chemically crosslined matrix (chemcial gel), radical polymerisation to cast, gel conc 3.5-20%, DNA size range 6-2,000 bp
5
Q
What is loaded into the gel other than the sample?
A
- Dye for visualisation of loading
- Dye for tracking electrophoresis
- Glycerol, glucose or urea for weighing sample (help it sink into well)
6
Q
What are two ways to visualise a gel?
A
- Fluorescent stain (Ethidium Bromide) - irrdiate with light to allow to visualise
- Visible stain (StainsAll) - more approachable
- These are intercalators - binds to the major grooves of DNA
7
Q
Capillary electrophoresis
A
- Can be used with many types of chemical and biochemical samples
- Fine capillary bridging between the two electrodes
- Electric field pushes the DNA through a capillary with gel inside it
- Obtains a 1D graph not a 2D image
- More controlled and calibrated - less variable that need to be controlled
8
Q
What causes smeared bands when visualising the gel?
A
isotropic release of radiation
9
Q
What is the difference in agarose and PAGE electrophoresis set up?
A
- agarose gel is flat
- PAGE gel is upright
10
Q
What is 32P labelling?
A
- expose to photographic film
- UV light will be absorbed by DNA so decreased transmission but will damage the DNA fast
11
Q
What is the lower band on the gels likely to be, and what mistakes might have been made to result in its occurance?
A
- formation of primer dimer
- due to poor choice of primer sequence or too much primer added
