Worksheet 10 - DNA Melting Curve
Course Code: BICH172
Course Name: Introductory Biochemical Laboratory
Student Name: CHUNG YUK MAN EMILY
Student ID: 10198465
Group Number: 10
Date of Experiment: 16th November 2010
Q1.State the experimental aim and describe the term “hyperchromic effect”. (2 mark)
The experimental aims are to study the basic structural characteristic of DNA molecules through UV spectrum and thermal denaturation and to understand how pH and ionic strength affect the stability of DNA which lead to the shift of melting temperature Tm. Hyerchromic effect is the increase of absorbance at 260nm during the double strand DNA is unwounded into two single strands DNA during denature.
Q2.Construct melting curves of DNA at different conditions on the same graph. (2 marks)
Q3.Define the term Tm. Describe the melting curve of DNA in TE buffer and indicate its Tm value. (2 marks)
Tm is the temperature at which half of the DNA strands are in the double-helical state and of them are in the "random-coil" state. It can be determined from the following equation,
Aaverage = (Amax + Amin) /2
The melting curve of DNA in TE buffer is in sigmoidal shape and can be divided into three parts. The first part of the curve (20oC-60oC) is flat and smooth. The absorbance value is low with little variation. It means that most DNAs remain in their double helix form and not being denatured. Then, there is a dramatic increase in the second part of the curve (60oC-80oC). It means that many DNA molecules undergo thermal denaturation. Lastly, the curve levels off again at relatively high absorbance values in the third part (80oC-100 oC). It means all of the double strands in DNA have denatured and been separated. From the graph, the Tm of DNA TE buffer should be about 75oC.
Q4.Explain how guanine-cytosine (GC) content of a DNA strand can affect its Tm value. (1...