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  Section: Biotechnology Methods » Molecular Biology
 
 
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Sucrose Density Fractionation

 
     
 
Content
Molecular Biology
  The Central Dogma
  Protein Synthesis in Cell Free Systems
  Chromosomes
  Polytene Chromosomes of Dipterans
  Salivary Gland Preparation (Squash Technique)
  Extraction of Chromatin
  Chromatin Electrophoresis
  Extraction and Electrophoresis of Histones
  Karyotype Analysis
  In Situ Hybridization
  Culturing Peripheral Blood Lymphocytes
  Microslide Preparation of Metaphases for In-Situ Hybridization
  Staining Chromosomes (G-Banding)
  Nucleic Acids
  Extraction of DNA from Bovine Spleen
  Purification of DNA
  Characterization of DNA
  DNA-Dische Diphenylamine Determination
  Melting Point Determination
  CsCl-Density Separation of DNA
  Phenol Extraction of rRNA (Rat liver)
  Spectrophotometric Analysis of rRNA
  Determination of Amount of RNA by the Orcinol Method
  Sucrose Density Fractionation
  Nucleotide Composition of RNA
  Isolation of Genomic DNA—DNA Extraction Procedure
  Isolation of Genomic DNA from Bacterial Cells
  Preparation of Genomic DNA from Bacteria
  Extraction of Genomic DNA from Plant Source
  Extraction of DNA from Goat Liver
  Isolation of Cotton Genomic DNA from Leaf Tissue
  Arabidopsis Thaliana DNA Isolation
  Plant DNA Extraction
  Phenol/Chloroform Extraction of DNA
  Ethanol Precipitation of DNA
  Isolation of Mitochondrial DNA
  Isolation of Chloroplast DNA
  DNA Extraction of Rhizobium (CsCl Method)
  Isolation of Plasmids
  RNA Isolation
  Preparation of Vanadyl-Ribonucleoside Complexes that Inhibit Ribonuclease Activity
  RNA Extraction Method for Cotton
  Isolation of RNA from Bacteroids
  Isolation of RNA from Free-Living Rhizobia
  Estimation of DNA purity and Quantification
  Fungal DNA Isolation
  Methylene Blue DNA Staining
  Transformation
  Blotting Techniques—Southern, Northern, Western Blotting
  Preparing the Probe
  Southern Blotting (First Method)
  Southern Blotting (Second Method)
  Western Blotting
  Western Blot Analysis of Epitoped-tagged Proteins using the Chemifluorescent Detection Method for Alkaline Phosphatase-conjugated Antibodies
  Southern Blot
  Southern Analysis of Mouse Toe/Tail DNA
  Northern Blotting
  Restriction Digestion Methods—Restriction Enzyme Digests
  Restriction Digestion of Plasmid, Cosmid, and Phage DNAs
  Manual Method of Restriction Digestion of Human DNA
  Preparation of High-Molecular-Weight Human DNA Restriction Fragments in Agarose Plugs
  Restriction Enzyme Digestion of DNA
  Electroelution of DNA Fragments from Agarose into Dialysis Tubing
  Isolation of Restriction Fragments from Agarose Gels by Collection onto DEAE Cellulose
  Ligation of Insert DNA to Vector DNA
  PCR Methods (Polymerase Chain Reaction)
  Polymerase Chain Reaction
  DNA Amplification by the PCR Method

Materials
  • 10% and 40% (w/v) Sucrose
  • 0.02 M sodium acetate, pH 5.1, containing 0.1 M NaCl and 1 mM
  • EDTA
    RNA
  • Ultracentrifuge, swinging bucket rotor, and tubes
  • Centrifuge tube-fractionating device
  • UV spectrophotometer
Procedure
  1. Form a 10–40% linear sucrose gradient in a nitrocellulose tube.
    1. Close all valves on the gradient device.
    2. Place exactly 15 mL of 40% sucrose in the right chamber and 15 mL of 10% sucrose in the left chamber of the gradient device.
    3. Open the flow from the right chamber to the centrifuge tube. Be sure there is a tube in place, that the flow is directed down the inside of the tube, and that the magnetic stirrer is functioning.
    4. Immediately open the valve on the left chamber and ensure that sucrose is flowing from left to right, thereby diluting the 40% sucrose with 10% sucrose.
    5. Allow the flow to continue until all of the sucrose enters the centrifugetube.
  2. Dissolve the RNA in 0.02 M sodium acetate solution to yield a final concentration of 250 µg/mL. The low pH of the solution helps to inhibit RNAse, while the salts will keep the RNA from forming large polymeric aggregates. Carefully layer 2.0 mL of the dissolved RNA onto the top of a sucrose gradient. This should be done by slowly allowing the solution to run down the side of the tube and onto the gradient. Be careful not to disturb the gradient.
  3. Load the ultracentrifuge with the prepared tubes and centrifuge for the equivalent of 18700 rpm for a Beckman SW27 rotor, for 20 hours at 4°C.
  4. At the completion of centrifugation, remove the tubes, and fractionate the contents into 1.0-mL fractions.
    1. Insert the nitrocellulose centrifuge tube into the plexiglass holder, but be careful not to puncture the bottom.
    2. Have a series of 30–35 test tubes ready to accept the effluent from the tubes. Each tube should be labeled and kept in order.
    3. Push down on the centrifuge tube (gently!) in order to puncture the bottom of the tube and immediately begin to collect the effluent.
    4. Count the drops that fall from the device, and place 40 drops into each tube.
  5. Using a UV spectrophotometer and microcuvettes, read the A260 of each fraction. Calculate the amount of RNA in each fraction.
  6. Plot the concentration of RNA in each fraction against the fraction number. Based on the density of sucrose in each fraction, compute the density of the RNA in that fraction. Based on the relative size (greater density) of the
    RNA, determine the nature of the fractions (i.e., rRNA, tRNA).
 
     
 
 
     




     
 
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