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 adjustment 2.Tip ejector button 3.Plunger 4.Adjustment threads 5.Volume indicator window 6.Tip ejector bracket 7.Shaft 8.Disposable tip

Pipette tips Different tips for different kinds of pipettes Micropipettes are never used without an appropriate tip

How micropipettors work Pipettes have two springs “First stop”: calibrated volume “Second stop”: maximum volume of pipette

What is the volume indicated?

6.84 uL132.5 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 volume C: Put tip into new tube and press plunger to SECOND STOP, expelling liquid D: Remove tip from liquid BEFORE releasing plunger E: 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 V s = settling velocity (m/s) r = radius of the particle (m), g = gravitational force (m/s 2 ) ρ p = particle density (g/mL) ρ f = 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 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 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. Parent cell First replication Second replication

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