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Micropipettes and Centrifuges. Bio 101A. Centrifugation. A means of purification of solids and liquids mixed in a suspension Done by varying applied force (gravity) A variety of uses in Biology. Centrifugation theory. *. V s = settling velocity (m/s) r = radius of the particle (m),

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centrifugation
Centrifugation
  • A means of purification of solids and liquids mixed in a suspension
  • Done by varying applied force (gravity)
  • A variety of uses in Biology
centrifugation theory
Centrifugation theory

*

  • Vs = settling velocity (m/s)
  • r = radius of the particle (m),
  • g = gravitational force (m/s2)
  • ρp = particle density (g/mL)
  • ρf = fluid density (g/mL)
  • μ = fluid viscosity (Pa s).
  • (*equation not on quiz…)
uses of centrifugation
Uses of centrifugation
  • Purifying cells
  • Purifying organelles
  • Purifying molecules
  • After centrifugation, solid particles form a pellet
  • Liquid is called the supernatant
  • Supernatant and pellet are then easily separated
isolating organelles by cell fractionation
Isolating Organelles by Cell Fractionation

Cell fractionation takes cells apart and separates the major organelles from one another

Ultracentrifuges fractionate cells into their component parts

Cell fractionation enables scientists to determine the functions of organelles

le 6 5a

LE 6-5a

Homogenization

Tissue

cells

Homogenate

Differential centrifugation

slide7
1000 g

(1000 times the

force of gravity)

10 min

Supernatant poured

into next tube

20,000 g

20 min

80,000 g

60 min

Pellet rich in

nuclei and

cellular debris

150,000 g

3 hr

Pellet rich in

mitochondria

(and chloro-

plasts if cells

are from a plant)

Pellet rich in

“microsomes”

(pieces of plasma

membranes and

cells’ internal

membranes)

Pellet rich in

ribosomes

how is dna replicated
How is DNA replicated?
  • It was expected, but not proven, that DNA was replicated semiconservatively
  • Competing models were the conservative model and the dispersive model
le 16 10
Second

replication

First

replication

Parent cell

Conservative model. The two parental strands reassociate after acting as templates for new strands, thus restoring the parental double helix.

LE 16-10

Semiconservative model. The two strands of the parental

molecule

separate, and each functions as a template for synthesis of a new, comple-mentary strand.

Dispersive model. Each strand of both daughter molecules contains

a mixture of

old and newly synthesized

DNA.

meselson stahl experiment
Meselson-Stahl experiment
  • They labeled the nucleotides of the old strands with a heavy isotope of nitrogen
  • The first replication produced a band of hybrid DNA, eliminating the conservative model
  • A second replication produced both light and hybrid DNA, eliminating the dispersive model and supporting the semiconservative model
le 16 11
Bacteria

cultured in

medium

containing

15N

Bacteria

transferred to

medium

containing

14N

Gradient Centrifugation

Less

dense

DNA sample

centrifuged

after 20 min

(after first

replication)

DNA sample

centrifuged

after 40 min

(after second

replication)

LE 16-11

More

dense

Second replication

First replication

Conservative

model

Semiconservative

model

Dispersive

model

micropipettors
Micropipettors
  • Essential for moving liquids from container to container
  • Can pipette very small volumes, very precisely
  • Ubiquitous in Biology laboratories
  • Come in a variety of sizes
  • All require special disposable tips
  • They are fragile and expensive
pipette tips
Pipette tips
  • Different tips for different kinds of pipettes
pipettes have two springs
Pipettes have two springs
  • First stop- calibrated volume
  • Second stop- maximum volume of pipette
regular pipette and bulb
Regular pipette and bulb
  • For larger volumes
micropipettes have different volumes
Micropipettes have different volumes

Volumes adjustable with dial

what is the volume indicated18
What is the volume indicated?

6.84 uL

132.5 uL

264 uL

pressing the tip ejector button
Pressing the tip ejector button
  • Note: tip ejector doesn’t always work with certain tips