Centrifugation Flashcards
Structure of a centrifuge [7 marks]
- Refrigeration unit
- Rotor
- Motor
- Armoured locking lid
- Armoured bowl
- Driveshaft
- Motor
Types of roto [2 marks]
- Swing out: hinged, g force is aimed outwards, speed of spinning depends on strength of hinges.
- Fixed angle: tube is to an angle to g force, stronger than swing out rotors.
Types of centrifuge [4 marks]
- Benchtop centrifuge: separates whole cells, up to 3000rpm
- Benchtop microcentrifuge: purifies DNA/RNA, up to 20,000rpm
- High speed centrifuge: subcellular fractionation, up to 20,000
- Ultracentrifuge: subcellular franctionation, nucleic acid purification, up to 80,000rpm
What does the rate of sedimentation depend on? [3 marks]
- Centrifugal force (drives particle to the bottom of the tube)
- Buoyant density (present in fluid, opposes centrifugal force)
- Frictional drag (slows down particle before going to the bottom of the tube)
Force equation
Force = mass x acceleration
Acceleration equation
Acceleration = (rate of spin)^2 x distance from axis
What can be used to find RCF, rpm and rotating radius? [1 mark]
Nomograph
What is the sedimentation rate used for and what are its units? [2 marks]
- Measures size of a macromolecule
- Svedburg units (S)
Why do you need to balance a centrifuge? [1 mark]
To prevent damage to the rotor.
What can you do to open up mammalian cells? [3 marks]
- Mechanical stress in a homogeniser
- Ultrasonication
- Mild detergent treatment
How can nucleic acids be purified? [1 mark]
- Ethanol precipitation
How to distinguish between linear and plasmid DNA? [2 marks]
- Use CsCl
- Linear DNA binds more CsCl, plasmid DNA binds less.
What is one stain that can be used for DNA? [2 marks]
- Planar EtBr
- Intercalates between base pairs
