Melting curve - When absorbance rises the rise is steep...

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Melting curve . If you measure the absorbance of a sample under increasing denaturing conditions (i.e. heat) you can generate a melting curve ( Fig. 1 ). For example, lets assume that for a particular sample of DNA: Absorbance of double stranded DNA =1.00 Absorbance of single stranded DNA=1.37 Tm=melting temperature or the temperature at which the rise in A260 is half complete Notice from the curve: Absorbance remains constant up to temperatures well above those encountered by living cells in nature.
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Unformatted text preview: When absorbance rises, the rise is steep over 6-8C. Curve plateaus as the 2 strands separate. If you hold DNA length constant and the concentration of DNA in the sample constant, the Tm is a pretty good indication of base content. RULE: Tm increases as [G+C] increases . The higher the GC content of the DNA the higher the Tm. GC base pairing involves 3 hydrogen bonds (vs two in AT pairing) Therefore it takes more heat to disrupt the pairing....
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