- UV spectrophotometer (preferably with temperature control)
Dissolve your DNA preparation in SSC to produce a final concentration of
approximately 20 µg DNA/mL.
- Place the dissolved DNA in an appropriate quartz cuvette along with a
second cuvette containing SSC as a blank.
- 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.
- 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.
- Raise the temperature of the bath to 50°C and repeat step (a).
- Raise the temperature sequentially to 60°C, 65°C, 70°C, 75°C, and
80°C and repeat the absorbance measurements.
- 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.
- Correct all of the absorbance readings for solvent expansion relative to
- List the corrected values as At.
- 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
- Calculate the GC content of your sample using the formula
Percent of G + C = k(Tm – 69.3) × 2.44
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).