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  Section: Biotechnology Methods » Molecular Biology
 
 
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Phenol Extraction of rRNA (Rat liver)

 
     
 
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
  • Rat liver (fasted rat)
  • Liquid nitrogen
  • p-Amino-salicic acid
  • Phenol mixture
  • Homogenizer or blender
  • Refrigerated preparative centrifuge
  • NaCl
  • 95% and 70% (v/v) ethanol
Procedure
  1. Obtain a rat that has been fasted for 24 hours (to remove glycogen from the liver), decaptitate, exsanguinate, and remove the liver as rapidly as possible.
  2. Weigh the liver don’t allow it to dehydrate.
  3. Immediately drop the liver into a container of liquid nitrogen.
    Caution:
    Liquid nitrogen will cause severe frostbite!
  4. Using the weight of the liver as an indication of the volume (1 gm of liver equivalent to 1 mL), add 15 volumes of freshly prepared 6% para-aminosalicylate (pAS) to a chilled blender or homogenizer.
  5. Add an equal volume (equal to the pAS) of phenol mixture to the blender and turn on the blender for a short burst to mix the pAS and phenol.
    Caution: Phenol is extremely caustic. Phenol causes severe skin burns, yet it is a local anesthetic. You will be unaware of the burn at first, except for telltale discoloration of the skin and blisters. You will become aware of the burn as the anesthetic properties wear off. Phenol also readily dissolves most countertops and all rubber compounds.
  6. Stop the blender and add the frozen liver (handle the liver with long forceps, or tongs). Blend the entire mixture (pAS, phenol, and liver) for 30 seconds at full speed. Do not blend for longer periods or you will sheer the RNA.
  7. Carefully transfer the homogenate to a beaker and continue to stir the mixture for 10 minutes at room temperature.
  8. Transfer the homogenate to nalgene centrifuge tubes and centrifuge the mixture at 15600 xg at 4°C for 20 minutes.
  9. Remove the centrifuge tubes and carefully separate the upper aqueous layer from the lower phenol layer. Take care that none of the white interphase material is mixed into the aqueous layer. The upper layer can most efficiently be removed by using a large hypodermic equipped with a long, large-bore, square-tipped needle. Should some of the interphase material be stirred into the aqueous phase, it will be necessary to repeat step 8.
  10. Measure the volume of the aqueous layer and discard the phenol layer and interphase material.
  11. Add 3.0 grams of NaCl per 100 mL of aqueous phase and stir until dissolved.
  12. Add 0.5 volumes of phenol mixture to the aqueous phase, place into a suitable flask, and shake vigorously for about 5 minutes. Recentrifuge as in step 8 above, but for 10 minutes.
  13. Separate the aqueous phase and add 2 to 3 volumes of cold 95% ethanol. Allow the mixture to stand in the freezer until a precipitate forms.
  14. Collect the RNA precipitate by centrifugation, wash once in 70% ethanol and store in 70% ethanol at 0–5°C.
Notes
Knowledge of transcription is based on our ability to extract “native” or functional RNA molecules from cells, with subsequent use of those molecules “in vitro.” One of the earliest methods for this type of analysis is a phenol-detergent extraction of RNA coupled with separation of the various-sized molecules of RNA with centrifugation in a gradient.
This basic procedure remains useful today, although there have been myriad additions and alterations to the procedure using a host of extraction techniques and separation procedures (such as electrophoresis or column chromatography).
 
     
 
 
     




     
 
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