Exercise 14.5 - Melting Point Determination

LEVEL II

Materials

• DNA
  
• SSC
  
• UV spectrophotometer (preferably with temperature control)

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:

   a. 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.

   b. Raise the temperature of the bath to 50° C and repeat step a.

   c. Raise the temperature sequentially to 60° C, 65° C, 70° C, 75° C and 80° C and repeat the absorbance measurements.

   d. Slowly raise the temperature above 80° 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.

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

5. Plot the value of A /A 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 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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