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To calculate the Extinction coefficient, an approach proposed by Richard Owczarzy is used: http://www.owczarzy.net/extinctionDNA.htm. That is for a single-stranded molecule:
Extinction coefficient = sum(extinction coefficients of all dinucleotides) - sum(extinction coefficients of inner mononucleotides) |
For example, let's calculate the molar extinction coefficient ("ε") for "ATGCA":
ε(ATGCA) = ε(AT) + ε(TG) + ε(GC) + ε(CA) - ε(T) - ε(G) - ε(C) = = 22800 + 19000 + 17600 + 21200 - 8700 - 11500 - 7400 = = 53000 |
As for the other statistics, average values are used in case of degenerate base characters.
Extinction coefficientfor a double-stranded molecule is calculated as a sum of the extinction coefficients of the two single strands (es1 + es2) multiplied by coefficient of (1 - hypochromicity h260nm). The hypochromicity effect can be taken into account account as follows:
h260nm = (0.287fAT + 0.059fGC) |
where fAT and fGC are fractions of AT and GC base pairs, respectively.
Melting temperature
The melting temperature is calculated as follows. For sequences of length 15 or longer:
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nmole/OD260
The amount of DNA of or RNA represented in nanomoles per 1 unit of absorbance at 260 nm dissolved in 1 ml cuvette with 1 cm pathlength.
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μg/OD260
The amount of DNA of or RNA represented in microgrames per 1 unit of absorbance at 260 nm dissolved in 1 ml cuvette with 1 cm pathlength.
μg/OD260 = nmoleOD260 * molarWeight * 0.001 |
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