Micropipettes and Centrifuges

Bio 9

Micropipettes are essential equipment in a modern biology lab

• For moving liquids from container to container

• Can measure tiny volumes, very precisely

• Come in a variety of sizes

• All require special disposable tips

• They are fragile and expensive

Micropipettes have different volumes

• P20: 20 microliters (=20 uL) max vol.• P200: 200 uL max.• P1000: ____uL max vol = ______

Volumes adjustable with dial

Micropipettes have different volumes

• P20: 20 microliters (=20 uL) max vol.• P200: 200 uL max.• P1000: __1000uL max vol = _1mL__

Volumes adjustable with dial

Parts of a mircopipette

1. Volume adjustment2. Tip ejector button3. Plunger4. Adjustment threads5. Volume indicator

window6. Tip ejector bracket7. Shaft8. Disposable tip

Pipette tips

• Different tips for different kinds of pipettes

• Micropipettes are never used without an appropriate tip

How micropipettors workPipettes have

two springs“First stop”:

calibrated volume

“Second stop”: maximum volume of pipette

What is the volume indicated?

What is the volume indicated?

6.84 uL 132.5 uL 264 uL=____mL

How to use the pipette

A: Press plunger down to FIRST STOP BEFORE putting in liquid (make sure you adjusted to the right volume!)

B: Release plunger slowly, taking up correct volumeC: Put tip into new tube and press plunger to SECOND

STOP, expelling liquidD: Remove tip from liquid BEFORE releasing plungerE: Release plunger and eject tip

How to take care of the pipettes

• Never use a pipette without a tip• Never adjust a pipettor beyond its maximum (or

minimum) volume range• Never place a pipet on its side with liquid in the tip

What is wrong with this picture?

Pressing the tip ejector button

• Note: tip ejector doesn’t always work with certain tips

Centrifuges

Centrifugation

• A means of purification of solids and liquids mixed in a suspension

• Done by varying applied force (gravity) f = rN^2

• A variety of uses in Biology

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

• 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

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

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

Semiconservative model. The two strands of the parental moleculeseparate, 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.

Parent cellFirstreplication

Secondreplication

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