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  Section: Biotechnology Methods » Molecular Biology
 
 
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Nucleotide Composition of RNA

 
     
 
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
  • RNA sample
  • 1 N and 0.1 N HCl
  • Boiling water bath
  • Whatman #1 filter paper (for chromatography)
  • Chromatography tank
  • 20-µL micropipette
  • Acetic acid:butanol:water (15:60:25) solvent
  • UV light source
  • UV spectrophotometer
Procedure
  1. Place a portion of your RNA sample (approximately 40 mg, hydrated) into a heavy-walled pyrex test tube. Add 1.0 mL of 1 N HCl and seal the tube.
  2. Heat the tube in a boiling water bath for 1 hour.
  3. Cool the tube, open it, and place the contents into a centrifuge tube. Centrifuge the contents at 2000 rpm in a clinical centrifuge to remove any insoluble residue. The supernatant contains your hydrolyzed RNA.
  4. Prepare Whatman filter paper No. 1 for standard one-dimensional chromatography.
  5. Using a micropipette, spot 20 µL of your hydrolyzate onto the paper, being careful to keep the spots as small as possible (repeated small drops are better than 1 large drop). Allow the spots to completely dry before proceeding.
  6. Place the paper chromatogram into your chromatography tank and add the solvent (acetic acid:butanol:water). Allow the system to function for an appropriate time (approximately 36 hours for a 20-cm descending strip of Whatman #1). Remove the paper and dry it in a circulating air oven at 40°C for about 2 hours.
  7. Locate the spots of nucleotides by their fluorescence under an ultraviolet (UV) light source. Expose the paper chromatogram to a UV light source and outline the spots using a light pencil. The order of migration from the point of origin is guanine (light blue fluorescence), adenine, cytilic acid, and finally, uridylic acid. Do not look directly at the UV light source. Use a cabinet designed to shield from harmful UV radiation.
  8. After carefully marking the spots, cut them out with scissors and place the paper cutouts into separately labeled 15-mL conical centrifuge tubes. Add 5.0 mL of 0.1 N HCl to each tube and allow the tubes to sit for several hours to elute the nucleotides from the paper.
  9. Pack down the paper with a glass rod (centrifuge in a clinical centrifuge if necessary) and remove an aliquot of the liquid for spectrophotometric assay.
  10. Measure the absorbance of each of the four nucleotides at the indicated UV wavelength (having first blanked the instrument with 0.1 N HCl).


  11. Use the molar extinction coefficients to determine the concentration of each base in the sample. Calculate the percent composition of each base, and the purine/pyrimidine ratio.
 
     
 
 
     




     
 
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