Handling Electrophoresis Flashcards
1
Q
Factors affecting stability
A
- Sequence/length
- Concentration
- Temperature
- Salts
- pH
- Denaturants
2
Q
What wavelengths do nucleobases absorb at
A
The nucleobases absorb at 260 nm due to their aromatic rings
3
Q
Electrophoresis theory
A
- Used to separate DNA
- DNA is placed ina buffer solution with a cathode and anode
- As DNA is negatively charged it migrates towards the postively charged anode.
4
Q
Electrophoresis theory
Movement rate of DNA
A
- Movement is proportional to charge/mass ratio
- A 10mer and 100mer will migrate at the same speed.
- No matter the length, the mass to charge ratio will always be the same so it will migrate at the same rate in solution
5
Q
How do we separate different lengths of DNA?
A
- Pass them through a selective medium: gel
- The gel has intertangled fibrils and a trapped solvent.
6
Q
Electrophoretic separation
Small vs large fragments
A
- Fibrils hinder the movement of larger molecules
- Gel electrophoresis of nucleic acids separates by size and shape, since mass/charge ratio is constant
- The same amount of DNA, if compacted, will move faster than a rigid rod double helix
- Small fragments pass through quickly
- Larger molecules, the fibrils interrupt it so they don’t make it all the way through the gel.
7
Q
Electrophoretic separation
Electrodes
A
- Samples get loaded at the cathode end.
- Using a ‘ladder’ lets you compare your sample with known lengths.
- Migration distances can be calibrated to make accurate estimates of intermediate lengths.
- Shorter strands move faster, and therefore appear at the bottom of the gel.
- Larger base pairs sit at the top.
8
Q
Types of gel
A
- Agarose
- Poly(acrylamide) / PAGE
9
Q
Poly(acrylamide)
PAGE
A
- Chemical gel
- Chemically crosslinked matrix
- 3.5-20% gel concentration
- 6-2000 bp DNA size range
- Long polymer with side chains which creates a controlled matrix
- Polymerisation turns it into a gelatinous material
- Used for short amounts
- Concentration and density of space matters
- Can be native or denatured
- Cast through radical polymerisation
10
Q
Agarose
A
- Physical gel
- Tangled fibrils
- 0.5-2.0% gel concentration
- 50-30,000 bp DNA size range
- Used for larger DNA
- Concentration and density of space matters
11
Q
Loading a gel
A
- Dye for visulisation of loading
- Dye for tracking electrophoresis
- Glycerol, glucose or urea for weighting sample (makes it more dense than the buffer solution) - without weighing it down it wouldn’t sit at the bottom of the well
- Its possible a long chain of DNA has been cut up to look at the base pairs.
12
Q
Visualising a gel
UV light
A
- DNA absorbs UV light so decreased transmission can be observed but UV light damages DNA within 15 seconds
13
Q
What does smeared bands mean in electrophoresis?
A
isotropic release of radiation
14
Q
How does fluorescent staining work?
A
- Intercalators bind to the major groove of DNA
- They are often carcinogenic
15
Q
A
16
Q
How to make nucleic acids?
A
- Chemially synthesis nucleotides in a controlled environment to form oligonucleotides
- Enzymatically synthesis oligonucleotides to get polynucleotides/genes
17
Q
Oglionucleotides
A
<200 bases
18
Q
Polynucleotides/genes
A
~1000-4000 bases
19
Q
Chemical synthesis disconnections
A
- Usually bonds break between carbon and other atoms
- Phosphate linker is a good site since then each base can be added step by step
- Phosphorous chemistry is vital in order to control the synthetic pathway
20
Q
Solid phase synthesis of oligonucleotides
A
- To perform each step in solution would require a purification step at each stage.
- By attaching one end of the strand to a solid support, the excess reagents and side products can simply be washed away.
- Introduce a gene to a glass bead one at a time until we have a set of what our primary structure wants to be.
- The glass bead has cavities to maximise the surface area
- It is then cut off and we have our desired nucleic structure.
