biochrom HB Ultrospec 7500, Руководство пользователя, Страница: 94 / 104

Ultrospec 7500 User Manual • 41 56 2050 REV03 • 94
Melting Temperature (T
m
)
T
m
(°C) =
ΔH
(16.6 × log
10
[Buffer]) + ( α + ΔS + (R × ln[c ÷ 4])) - 273.15
ΔH is the change in enthalpy (kcal mol
-1
) and ΔS is the change in entropy (kcal K
-1
mol
-1
), and are the sum values of their nearest-neighbour
pair values, specifically:
Molecule DNA RNA
Pair* ΔH ΔS ΔH ΔS
AA:TT/UU -9.1 -0.0240 -6.6 -0.0184
AT/AU:TA/UA -8.6 -0.0239 -5.7 -0.0155
TA/UA:AT/AU -6.0 -0.0169 -8.1 -0.0226
CA:GT/GU -5.8 -0.0129 -10.5 -0.0278
GT/GU:CA -6.5 -0.0173 -10.2 -0.0262
CT/CU:CG -7.8 -0.0208 -7.6 -0.0192
GA:CT/CU -5.6 -0.0135 -13.3 -0.0355
CG:GC -11.9 -0.0278 -8.0 -0.0194
GC:CG -11.1 -0.0267 -14.2 -0.0349
GG:CC -11.0 -0.0266 -12.2 -0.0297
* The nucleotide pair on the left of the colon is the 5’ to 3’ sequence while the nucleotide pair on the right of the colon is the 3’ to 5’
sequence.
The nucleotide pair on the left of the forward-slash is the DNA sequence while the nucleotide pair on the right of the forward-slash is
the RNA sequence.
Buffer is the buffer concentration (M).
α is the helix initiation factor fixed at -0.0108 kcal K -1 mol -1
.
R is the gas constant fixed at 0.001987 kcal K -1 mol -1 .
c is the calculated nucleic acid concentration (M), specifically:
c = A260 × Calculated Factor
A260 is the absorbance at 260 nm.
Calculated Factor is calculated from the molecular weight and molar extinction coefficient (E).
Calculated Factor =
Molecular Weight
Molar Extinction Coefficient
Molecular Weight is calculated for the base sequence defined within the application method parameters.
Molecular Weight of DNA = 312.2 × number of A + 288.2 × number of C + 328.2 × number of G + 303.2 × number of T + K
Molecular Weight of RNA = 312.2 × number of A + 288.2 × number of C + 328.2 × number of G + 289.2 × number of U + K
K is phosphorylation constant, specifically
K
= 17 + (N+2) × Counter Ion
(Phosphorylated)
K
= -61 + (N+1)
× Counter Ion
(Non-phosphorylated)
N is the base sequence length
Counter ion is the molecular weight of the counter ion selected: