Centrifugation Flashcards
What is benchtop centrifuge used for?
Used to separate whole cells
What is typical speed of benchtop centrifuge?
Few hundred to 3000 rpm
What is high speed centrifuge used for?
Used for subcellular fractionation
Used to separate organelles
What is typical speed for high speed centrifuge?
Up to 20,000 rpm
What is microcentrifuge used for?
Used to purify DNA + RNA
What is typical speed of micro centrifuge ?
Up to 20,000 rpm
What is ultracentrifuge used for?
Used for nucleic acid purification
Used for subcellular fractionation
What does rate of sedimentation of particle depends on?
Centrifugal force
Buoyant density
Frictional drag
More massive the particle, the …… centrifuge force so …….it will sediment
Bigger
Faster
For given mass, the more ….. the particle the ….. it will sediment=> more ……so….. drag therefore ….. buoyancy
Dense Faster Compact Less Less
Centrifugal force depends on mass of particle + acceleration
What is the equation for force? And what is its units?
Force= mass x acceleration
Force is measured in Newtons
Acceleration depends on distance from axis and the rate of spin squared
What is the equation for acceleration?
Acceleration= w^2r
w=rate of spin
r= distance from axis
What is typical speed for ultracentrifuge?
Up to 80,000 rpm
Sedimentation rate is measured in …….
Svedberg units
What are some practical points of centrifuge?
Sample in centrifuge must be balanced
Every tube must have another directly opposite
Tubes must contain same volume of fluid
Low speed centrifuge + microcentrifuge=> balance tubes by eye
High speed centrifuge + ultracentrifuge=> balance tubes by weighing them
What happens if mammalian cells are centrifuged at high speeds?
Cells may lyse due to shear forces
Mammalian cells can be broken open by:
Mechanical stress in homogeniser
Ultrasonication
Mild detergent treatment
How can you purify nucleic acids?
Add NaCl solution
Add ethanol to final concentration of 70%
Centrifuge at 5-10,000g
Rinse pellet in 70% ethanol + spin again
Remove supernatant
Allow pellet to air dry
What does isopynic mean?
Means equal density
Nucleic acids can be purified and separated by ……… in ………
Isopynic centrifugation in caesium chloride density
