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and displays the results. The ratio of the Absorbance values at wavelengths 1 and
2 are calculated. The Concentration is based on the Absorbance at wavelength 1.
Repeat step 9 for all samples.
Press
Esc
to return to the Life Science Screen.
Press
to display available Options which are described below.
Options (select using keypad numbers)
1. Return to Parameters Screen (step 1 above).
2. Print result via selected method.
3. Toggle Graph on/off. The graph shows a Wavescan plot across the range
220 nm to 320 nm with cursors denoting 230, 260, 280 and (if Background
Correction is selected) 320 nm.
7. Sample Number – add a prefix to the sample number and reset the
incrementing number to the desired value.
8. Save Method – use the alpha-numeric keys to enter a name for the method and
press Save
.
9. Auto-Print – toggles Auto-Print on/off.
Exit options by pressing
Esc
, or wait.
4.2.5. T
m
Calculation
This utility calculates the theoretical melting point from the base sequence of a
primer. It is done using nearest neighbor thermodynamic data for each base in the
nucleotide chain in relation to its neighbor (Breslauer et al, Proc.Natl. Acad. Sci. USA
Vol 83, p3746 1986). The data obtained are useful both in the characterization of
oligonucleotides and in calculating T
m
for primers used in PCR experiments.
The ACGT/U sequence entered into the utility is used to calculate the theoretical
T
m
, the theoretical Absorbance (Absorption units/mmol) and the conversion factor
(mg/ml). This is because the stability of a bent and twisted sequence of bases such
as an oligonucleotide is dependent on the actual base sequence. The calculated
thermodynamic interactions between adjacent base pairs have been shown to
correlate well with experimental observations.
This utility uses matrices of known, published thermodynamic values and extinction
coefficients to calculate T
m
and the theoretical Absorbance/factor respectively of an
entered base sequence.
T
m
This is calculated using the equation:-
T
m
= ∆H × 100
- 273.15 +16.6 log [salt]
∆S + (1.987 × log
e
(
c/4 +53.0822)
where
∆H and ∆S are the enthalpy and entropy values summed from
respective 2 × 4 × 4 nearest neighbor matrices
c is the Primer concentration of oligonucleotide (pmoles/ml) in the
calculated T
m
or the measured concentration in measured T
m
.
In the latter case concentration is obtained from the equation:
c =
Abs(260 nm) × Calculated factor × pathlength multiplier × 10 000
MW