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DNA is generally stable than RNA, but there are many chemical and physical factors
affect the nucleic acids. The chemical factors that affect nucleic acids are such as hydrolysis
by acids, alkali, enzymes, and mutagenic factors of the DNA bases. The physical factors are
heat, pH, salt concentration, and base composition.
Strand separation,
denaturation, or
melting
dsDNA
Helix
ssDNA
Annealing,
renaturation, or
hyberdization
Coil
Slowly cool
Boiled
15 min
dsDNA
Rapidly cool
Material:
Deoxyribonucleic acid solution
Ice
Method:
Place the solutions (5 ml) in cuvette in a spectrophotometer and record the absorbance at room
temperature. Use distilled water as blank in this experiment.
Transfer the DNA solution to test tube with thermometer and place in water bath, then record
the change in UV absorption every 10C elevation in temperature.
Note: The temperature of the water bath is usually higher than the temperature of the test tube
contents, so the temperature of the DNA solutions should be measured directly if possible.
Every time record the temperature and absorbance and continue until no change in absorbance
is recorded (around 95C.)
Slowly cool the DNA solution from 95C until room temperature and measure the extension
every 10C decrease.
Repeat the experiment but this time rapidly cools the solution in ice and record the change in
absorbance every 10C decrease.
Result sheet
3. Construct DNA rapid cooling curve and other group construct DNA slow cooling curve by
drawing a graph using the absorbance readings against the temperature for each
experiment.
4. Compare the cooling and melting curves for the two experiments and comment on the effect
of temperature.
Absorbance
Temp. of rapid
cooling on ice
95C
25C
Absorbance
Absorbance
Useful links:
http://www.chemistry.nmsu.edu/studntres/chem435/Lab4/intro.html
http://oregonstate.edu/instruction/bb492/lectures/StructureI.html