Exercise 14.5 - Melting Point Determination

LEVEL II

Materials

Procedure 4

  1. Dissolve your DNA preparation in SSC to give a final concentration of approximately 20 µg DNA/ml.

  2. Place the dissolved DNA in an appropriate quartz cuvette along with a second cuvette containing SSC as a blank.

  3. 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:

  4. Correct all of the absorbance readings for solvent expansion relative to 25° C. 5 List the corrected values as A_t

  5. Plot the value of A_t /A_2_5 vs 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) x 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 three hydrogen bonds as opposed to the two 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 UV_2_6_0 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° and 100° C in this buffer (as opposed to 25° C in distilled water).

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Cell Biology Laboratory Manual
Dr. William H. Heidcamp, Biology Department, Gustavus Adolphus College,
St. Peter, MN 56082 -- cellab@gac.edu