filtering and centrifugation physical separation of solids from liquids
Post on 22-Dec-2015
216 views
TRANSCRIPT
Filtering and Centrifugation
Physical Separation of Solids from Liquids
Part I – FiltrationFamiliar filtering - funneling
Paper filters with simple funnels
Buchner Funnels Bacteria, fungi,
viruses pass through easily
Vacuum filtration
Replaceable Membranes
Membranes must be appropriate pore size
Bacteria > 0.3 m Viruses > 0.02 m
(not filterable)
Depth Filter
Asbestos or glass fibers.
Tortuous path, particles trapped in filter
Clarifying solutions
Membrane filter
Highly polymerized nitrocellulose or polysulfone
Pore size controlled by polymerization reaction
Particles (bacteria, fungi) trapped on surface, some in filter
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
Like this
Disposable filter units
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
Part II – Centrifuges, rotors, and their tubes
Centrifugal force
))()(1012.1( 25 rxF 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
RCF as a function rpm
0
20000
40000
60000
80000
100000
120000
0 5000 10000 15000 20000 25000 30000
rpm
RC
F
15 cm
7 cm
3 cm
Pellets and supernatants from cultures
Supernatant – usually spent media to be discarded.
Pellet – bacterial or yeast cells to be collected
Pellets and supernatants from cell lysis studies
Supernatant – may contain DNA or other liberated cell constiituent.
Pellet – Cell debris to be discarded
Pellets and supernatants from DNA precipitation
Supernatant – alcohol and salt used to precipitate DNA
DNA Pellet – Warning! DNA pellets are pretty much invisible
Minifuges
14,500 rpm or 14,000 x g
Pellet bacteria Economical, small
foot print
Microfuges
13,000 rpm or 16,000 x g
More samples, sturdier
Pellet bacteria, can collect DNA
Tabletop centrifuges
>20,000 rpm or >35,000 x g
Widest applications Similar to Avanti Refrigerated units
preferred to collect DNA
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
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
Conical tubes
Pre-sterilized, plastic disposable
Maximum force of only 6,000 -9,000 x g
Not compatible with solvents!
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
Place your tubes in the rotor
Tubes of equal mass opposite one another
Hinges up
Ready to try?