Centrifugation

Question 1

What is benchtop centrifuge used for?

Answer 1

Used to separate whole cells

Question 2

What is typical speed of benchtop centrifuge?

Answer 2

Few hundred to 3000 rpm

Question 3

What is high speed centrifuge used for?

Answer 3

Used for subcellular fractionation

Used to separate organelles

Question 4

What is typical speed for high speed centrifuge?

Answer 4

Up to 20,000 rpm

Question 5

What is microcentrifuge used for?

Answer 5

Used to purify DNA + RNA

Question 6

What is typical speed of micro centrifuge ?

Answer 6

Up to 20,000 rpm

Question 7

What is ultracentrifuge used for?

Answer 7

Used for nucleic acid purification

Used for subcellular fractionation

Question 8

What does rate of sedimentation of particle depends on?

Answer 8

Centrifugal force
Buoyant density
Frictional drag

Question 9

More massive the particle, the …… centrifuge force so …….it will sediment

Answer 9

Bigger

Faster

Question 10

For given mass, the more ….. the particle the ….. it will sediment=> more ……so….. drag therefore ….. buoyancy

Answer 10

Dense 
Faster 
Compact 
Less 
Less

Question 11

Centrifugal force depends on mass of particle + acceleration
What is the equation for force? And what is its units?

Answer 11

Force= mass x acceleration

Force is measured in Newtons

Question 12

Acceleration depends on distance from axis and the rate of spin squared
What is the equation for acceleration?

Answer 12

Acceleration= w^2r
w=rate of spin
r= distance from axis

Question 13

What is typical speed for ultracentrifuge?

Answer 13

Up to 80,000 rpm

Question 14

Sedimentation rate is measured in …….

Answer 14

Svedberg units

Question 15

What are some practical points of centrifuge?

Answer 15

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

Question 16

What happens if mammalian cells are centrifuged at high speeds?

Answer 16

Cells may lyse due to shear forces

Question 17

Mammalian cells can be broken open by:

Answer 17

Mechanical stress in homogeniser
Ultrasonication
Mild detergent treatment

Question 18

How can you purify nucleic acids?

Answer 18

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

Question 19

What does isopynic mean?

Answer 19

Means equal density

Question 20

Nucleic acids can be purified and separated by ……… in ………

Answer 20

Isopynic centrifugation in caesium chloride density