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  Section: Biotechnology Methods » Molecular Biology
 
 
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DNA-Dische Diphenylamine Determination

 
     
 
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
  • Lyophilized DNA standard
  • Sample DNA
  • SSC
  • Dische diphenylamine reagent
  • Spectrophotometer

Procedure
  1. Weigh out 15.0 mg of commercial lyophilized DNA and prepare a stock solution of 3.0 mg/mL by dissolving the DNA in 5.0 mL of SSC. This material will be used to prepare a standard curve for the diphenylamine reaction. Note that lyophilized, highly polymerized DNA is extremely slow to go into solution. It will require preparation at least 1 day in advance of the lab, with constant shaking.
  2. Prepare a series of known standard solutions by serially diluting the stock solution of DNA. Set up a series of test tubes containing 2.0 mL of SSC each. Pipette 2.0 mL of stock solution into tube #1, mix, pipette 2.0 mL of the resulting mixture into tube #2, and so on. This will yield a series of tubes containing 1.5, 0.75, and 0.375 mg/mL of DNA. Your original stock solution is 3.0 mg/mL and SSC should be used for the blank.
  3. Remove and discard 2.0 mL of the final dilution. To each of the 5 tubes in step 2 (each should contain only 2.0 mL), add exactly 4.0 mL of Dische diphenylamine reagent and mix well. This reagent contains glacial acetic acid. It is caustic and should be handled with care.
  4. Place a marble on the top of each test tube (it should not fall into the test tube, as it will act as a reflux to prevent evaporation, while allowing for pressure changes). Place the tubes in a boiling water bath for 10 minutes, remove from the bath, and immediately immerse in an ice bath to cool.
  5. Turn on a spectrophotometer and adjust the wavelength to 650 nm. Use the tube containing no DNA from step 2 to blank the instrument and measure the absorbance of each of your standards. Plot the absorbance against DNA concentration, perform a linear regression of the data, and compute the extinction coefficient.
  6. Dissolve your extracted or sample DNA in 10 mL of SSC. Make serial dilutions of 1/10, 1/100, and 1/1000 with SSC. Measure the absorbance of your extracted or sample DNA dilutions and calculate the concentration of DNA in the sample. Use the dilution which gives an absorbance in the 0.1 to 1.5 range

 
     
 
 
     




     
 
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