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  Section: Biotechnology Methods » Molecular Biology
 
 
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Melting Point 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
  • DNA
  • SSC
  • UV spectrophotometer (preferably with temperature control)

Procedure
Dissolve your DNA preparation in SSC to produce a final concentration of approximately 20 µg DNA/mL.
  1. Place the dissolved DNA in an appropriate quartz cuvette along with a second cuvette containing SSC as a blank.
  2. Place both cuvettes into a dual beam temperature regulated UV spectrophotometer and measure the absorbance of the sample at 260 nm at temperatures ranging from 25°C to 80°C. Continue to increase the temperature slowly and continue reading the absorbance until a sharp rise in absorbance is noted.
    Alternatively:
    1. Place the cuvettes into a waterbath at 25°C and allow to temperature
      equilibrate. Remove the blank, wipe the outside dry, and rapidly blank
      the instrument at 260 nm. Transfer the sample to the spectrophotometer
      (be sure to dry and work rapidly) and read the absorbance.
    2. Raise the temperature of the bath to 50°C and repeat step (a).
    3. Raise the temperature sequentially to 60°C, 65°C, 70°C, 75°C, and 80°C and repeat the absorbance measurements.
    4. Slowly raise the temperature above 80°C and make absorbance measurements every 2° until the absorbance begins to increase. At that point,increase the temperature, but continue to take readings at 1°C intervals.
  3. Correct all of the absorbance readings for solvent expansion relative to 25°C.
  4. List the corrected values as At.
  5. Plot the value of At /A25 versus temperature and calculate the midpoint of any increased absorbance. This midpoint is the melting point (Tm) for your DNA sample.
  6. Calculate the GC content of your sample using the formula
    Percent of G + C = k(Tm – 69.3) × 2.44
Notes
Single-strand DNA absorbs more UV light than double strands. Moreover, double strands can be separated by heat (melted) and the temperature at which the strands separate (Tm) is related to the number of guanine-cytosine residues (each having 3 hydrogen bonds, as opposed to the 2 in adenine-thymine). This has led to the development of a rapid test for an approximation of the GC/AT ratio using melting points and the change in UV260 absorbance (known as “hyperchromicity” or “hyperchromatic shift”). Of course, the separation is also dependent upon environmental influences, particularly the salt concentration of the DNA solution. To standardize this, all Tm measurements are made in SSC buffer. DNA melts between 85°C and 100°C in this buffer (as opposed to 25°C in distilled water).

 
     
 
 
     




     
 
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