Exercise 14.5 - Melting Point Determination
- UV spectrophotometer (preferably with temperature control)
- Dissolve your DNA preparation in SSC to give 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.
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
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
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.
- Correct all of the absorbance readings for solvent expansion
relative to 25° C. 5
List the corrected values as A
- Plot the value of A
vs temperature and calculate the midpoint of any increased
absorbance. This midpoint is the melting point (Tm) for your DNA
- Calculate the GC content of your sample using the formula
Percent of G + C = k(Tm -69.3) x 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 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
to Table of Contents
Cell Biology Laboratory Manual
Dr. William H. Heidcamp, Biology Department, Gustavus Adolphus College,
St. Peter, MN 56082 -- email@example.com