Materials:

Equipments:

1. 1. A UV/Visible spectrophotometer equipped with Peltier temperature cell:

Peltier accessory is used for achieving very high temperature accuracies. The temperature of the cells or cell holders can be monitored by placing temperature sensors. It is recommended to place the temperature sensors in the cells for achieving more accurate results.

2.  Water baths (in case equipment is not equipped with Peltier temperature control)

Glassware and plasticware:

1. 1. Pipettes

2. Pipette tips

3. Parafilm

4. 1.5 ml microfuge tubes

5. Distilled water

6. 1 ml Quartz cuvettes (Note 1)

7. The buffer the given DNA is dissolved in

Procedure:

1. 1.  Switch “ON” the spectrophotometer.

2.  Set the measurement mode to ‘Absorbance' and wavelength to 260 nm.

3. Set the temperature to 20°C in the Peltier attachment.

4. Measure the absorbance of the given DNA sample at 260 nm against the buffer used for dissolving the DNA.

5. An absorbance between 0.1 – 0.4 is suitable for determining the melting temperature. If the absorbance of the DNA is above 0.4, dilute the sample in the given buffer so as to achieve 1 ml DNA solution with an absorbance between 0.2 – 0.3.

6. Measure the absorbance of the diluted sample at 25°C.

7. Increase the temperature by 5°C and measure the absorbance when the cell reaches the specified temperature.

8. Repeat step 7 until a temperature of 95°C is achieved.

9. Record the measurements in the observation table shown below (Table 7.1).