Centrifugation

Ultracentrifugation

60,000 RPM and 200,000 x g Up to 150,000 RPM and 1,000,000 x g.

Preparative Centrifugation

A general procedure performed in preparation of further processing Often used to sediment cells or simple bring all liquid to the bottom of a shaken tube. "Spin down" / "Pellet Sample" / "Quick Spin"

Purification of Nucleic Acid

Cells and tissue are complex biochemical assemblages Many tissues (ex Bone) are difficult to breakdown and processing may degrade the DNA. Care must be taken not to introduce contaminants or degrade the sample

Cesium Chloride Density Gradient Centrifugation

Cells lysed using a detergent DNA is alcohol precipitated. DNA is mixed with CsCl & ethidium bromide (aids visualization) Centrifuged for several hours. High-quality DNA, yet time consuming and expensive.

Centrifugation

Centrifuges employ a spinning rotor contained in a stationary housing to apply centrifugal forces to a rotated sample. Given enough time, many particles in complex suspensions will settle out. Small particles (ex Viruses) may not settle unless unless subjected to very high G forces.

Rotor types

Swinging Bucket Fixed Angle Vertical Materials: Carbon fiber, aluminum, or titanium.

Sequential Differential Centrifugation

for separation of cells and sub-cellular components

Rotor Use and Care

Always Balance precisely your centrifuge tubes, particularly at high speeds. Never run you rotor above its maximum allowable rotor speed. Always make sure to set the centrifuge's control settings match to the rotor you intend to use! Always wash out your (metal fixed angle) Rotor and invert to dry on paper towels. Finally, make sure the tubes you intend to use are compatible!

Differential Centrifugation(General speed Guidelines)

1,000 x g / 10 minutes, intact cells and nuclei pellet to the bottom of the tube. 3,000 x g / 10 minutes used for bacteria. 10,000+ x g for 20 minutes to pellet subcellular organelles

Samples must be

Balanced EXACTLY

Organic Extraction

Conventional / older technique Organic solvents to extract contaminants from cell lysates. Cells are first lysed using a detergent Then mixed with phenol, chloroform, and isoamyl alcohol. Correct salt concentration and pH must be used during extraction to ensure that contaminants are separated into the organic phase and that DNA remains in aqueous DNA is usually recovered from the aqueous phase by alcohol precipitation.

Salting Out with Alcohol Precipitation

Each Component of the Lysis and Extraction Solution has at least 1 specific function: Sodium Docecyl Sulfate: Dissolves cell membranes: Sodium chloride: 1) Remove proteins bound to the DNA and 2) neutralizes charge repulsion between DNA strands, 3) keeps proteins dissolved in the aqueous layer so they don't precipitate in the alcohol along with the DNA. Proteinase K: Proteolytically degrades the protein yet leaves DNA intact. Finally, Isopropyl alcohol (Ice Cold!): Precipitates the DNA from solution. Strands of DNA can then be wrap around the glass rod near the interface between the two layers.

Density Gradient Centrifugation

Employed to purify macromolecules and /or subcellular organelles. Add layer on layer of a gradient medium (such as sucrose "cushion") in a tube with the heaviest layer at the bottom and the lightest at the top. Two categories: Rate-zonal (size) separation Isopycnic (density) separation.

Solid-phase anion-exchange chromatography

Exploits interaction between negatively charged phosphates of nucleic acid and positively charged surface molecules on the substrate resin. DNA binds under low-salt conditions while RNA, cellular proteins, and metabolites are washed out. High-quality DNA is eluted using a high-salt buffer. Eluted DNA is then recovered by alcohol precipitation.

Applications for Centrifugation

From simple spin down to sub cellular fractionation to analytical characterization of complexes. Can be used in conjunction with filter-sets to separate proteins based on Molecular Weight. Can be used to separate DNA and other nucleic acids Through specialized columns (ex. Silica columns)

Vertical Rotor Centrifuges

Highly Specialized & fairly rarely used Isopycnic separations* (ex. Cesium Chloride gradients)

Analytical Centrifugation

More sophisticated method: used to determine the physical characteristics of a sample such as a protein or protein complex. Allows their hydrodynamic and thermodynamic characterization in solution. Examples: Used to research dimer formation and other interactions like protein-protein, protein-nucleic acid, and protein-small molecule binding.

Fixed Angle Rotors

Most Commonly used type of rotor (WORKHORSE) Widely employed for differential separations (ie cell pelleting) Separate Cells (or cell debris) from supernatant Speeds vary from 10s all the way up to 1,000,000 × g (relative centrifugal force, RCF)!

K-factor

Pelleting efficiency of a rotor. (Smaller the K-factor, better the pelleting efficiency.)

tissue Disruption and Homogenization

Physical (Waring Blender) Sonication Grinding after liquid N2 flash Freezing Bead Beater with glass beads (1 mm) Osmotic Lysis (blood samples)

Swinging Bucket Rotors

Popularly used to separate large-volume samples (up to 12 L) at low speeds. High-throughput protocols like batch harvesting of whole cells from growth media. Processing of blood collection tubes, large-volume tissue culture processing, Even multiwell plates can be processed

DNA Isolation methods

Quick and Dirty (Colony PCR / crude extract ) Salting-out / Alcohol precipitation methods Organic extraction Cesium chloride density gradients Anion-exchange methods Silica-based methods

RCF

Relative Centrifugal Force (x gravity)

RPM

Rotations per Minute

S-value

Sedimentation coefficient describes molecular weight and shape of the particle, expressed in Svedberg units. The larger the S-value, the faster the particle separates. EX: RIBOSOMES 70S vs 80S

Organic extraction / Drawbacks

Time-consuming and labor intensive bio-technique. Toxic Chemicals used & Toxic Waste produced DNA can contain residual phenol and/or chloroform These can inhibit subsequent enzyme reactions Yield variability

Simplified DNA Isolation Protocol

Tissue Disruption Cell Lysis Removal of proteins and contaminants Recovery of DNA

Cell Lysis

Typically an ionic detergent (SDS) is used to destroy all cell membranes A proteinase like Proteinase K is used to destroy cellular proteins Physical methods may assist

Silica Gel Spin Columns

Utilizes specific adsorption of nucleic acids to a silica-gel membrane in the presence of high concentrations of certain salts Optimized buffers cause only DNA to be adsorbed while cellular proteins and other detritus are washed out. DNA is then eluted from the silica-gel membrane using a low-salt buffer. No alcohol precipitation is needed. Fast and easy and uses existing Centrifuges.