Micropipettes and Centrifuges

1. Micropipettes and Centrifuges Bio 101A

2. 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

3. 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…)

4. 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

5. 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

6. LE 6-5a Homogenization Tissue cells Homogenate Differential centrifugation

7. 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

8. 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

9. 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.

10. 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

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

12. 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

13. Pipette tips • Different tips for different kinds of pipettes

14. Pipettes have two springs • First stop- calibrated volume • Second stop- maximum volume of pipette

15. Regular pipette and bulb • For larger volumes

16. Micropipettes have different volumes Volumes adjustable with dial

17. What is the volume indicated?

18. What is the volume indicated? 6.84 uL 132.5 uL 264 uL

19. Never lay the micropipette on its side when liquid is in the tip (WHY NOT?)

20. Why is this bad?

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