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Filtering and Centrifugation

Filtering and Centrifugation

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Filtering and Centrifugation

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  1. Filtering and Centrifugation Physical Separation of Solids from Liquids

  2. Part I – FiltrationFamiliar filtering - funneling • Paper filters with simple funnels • Buchner Funnels • Bacteria, fungi, viruses pass through easily

  3. Vacuum filtration

  4. Replaceable Membranes • Membranes must be appropriate pore size • Bacteria > 0.3 m • Viruses > 0.02 m (not filterable)

  5. Depth Filter • Asbestos or glass fibers. • Tortuous path, particles trapped in filter • Clarifying solutions

  6. Membrane filter • Highly polymerized nitrocellulose or polysulfone • Pore size controlled by polymerization reaction • Particles (bacteria, fungi) trapped on surface, some in filter

  7. Nucleation track (Nucleopore) filters • Polycabonate films • Nuclear radiation and chemical etching cause holes in sheet • Typically sold in 0.2 and 0.45 m pores sizes • Particles trapped on surface

  8. Like this

  9. Disposable filter units

  10. Syringe filters • Disposable membrane or Nucleopore filters • Filter-sterilizing small volumes of liquids • Media, solutions, tissue culture • In line filters attach to tubing (pumps) • Also can be used for gasses

  11. Part II – Centrifuges, rotors, and their tubes

  12. Centrifugal force Force pressing the particle down relative to the force of gravity (RCF; units are g) Angular velocity expressed in rpm Radius, distance from center of rotation

  13. RCF as a function rpm 15 cm 7 cm 3 cm

  14. Pellets and supernatants from cultures Supernatant – usually spent media to be discarded. Pellet – bacterial or yeast cells to be collected

  15. Pellets and supernatants from cell lysis studies Supernatant – may contain DNA or other liberated cell constiituent. Pellet – Cell debris to be discarded

  16. Pellets and supernatants from DNA precipitation Supernatant – alcohol and salt used to precipitate DNA DNA Pellet – Warning! DNA pellets are pretty much invisible

  17. Minifuges • 14,500 rpm or 14,000 x g • Pellet bacteria • Economical, small foot print

  18. Microfuges • 13,000 rpm or 16,000 x g • More samples, sturdier • Pellet bacteria, can collect DNA

  19. Tabletop centrifuges • >20,000 rpm or >35,000 x g • Widest applications • Similar to Avanti • Refrigerated units preferred to collect DNA

  20. Ultracentrifuges • > 100,000 x g • Operate under vacuum – air creates heat from friction, and slows rotor down • Pellet membranes, ribosomes • Used in gradient work • CsCl – 24 hour separation of DNA • Sucrose – pelleting cell fractions small proteins to ribosomes • Svedberg Units – rate of migration through a sucrose gradient

  21. Rotors • Massive – stores kinetic energy • Fixed angle – Tubes held at about 45o angle to vertical • Swinging bucket – tubes on hinges. At full speed they go perpendicular to gravity

  22. Conical tubes • Pre-sterilized, plastic disposable • Maximum force of only 6,000 -9,000 x g • Not compatible with solvents!

  23. Microcentrifuge Tubes • Plastic, sterile, disposable centrifuge tubes • 2, 1.5, 0.5, and 0.2 (microamp) formats • Most molecular techniques, small reaction volumes • Special racks and storage

  24. Place your tubes in the rotor Hinges up Tubes of equal mass opposite one another

  25. Ready to try?