Techne Prime Elite Manuel d'utilisateur télécharger pdf (Page 24)

OLIGONUCLEOTIDE T

CALCULATOR

One of the most important parameters in a PCR is the annealing temperature of the reaction. This can

be determined theoretically using various software programmes to calculate the melting temperature

) of the primers.

The T

is defined as the temperature at which half the DNA strands are single stranded and half are

double stranded when base paired to a complimentary strand. Using annealing temperatures that are

well below the T

of the primers can result in mismatching, false priming and may lead to primer-dimer

artefacts. Annealing temperatures that are significantly higher than the primer T

may result in

reduced priming or prevent priming altogether.

The Prime Elite uses the Nearest-Neighbour method for calculating the T

of an oligonucleotide

The T

is calculated using the following equation:

{(

∆H

º x 1000)/(A +

∆S

º + R ln(C/4))} - 273.15 + 16.6 x log[Na

]

Where:

∆H

= The sum of the Nearest-Neighbour enthalpy changes (Kcal/mol)

A = Helix initiation correction constant

∆S

= The sum of the Nearest-Neighbour entropy changes (cal K

mol

)

R = The Gas Constant (1.99 cal K

mol

)

C = Concentration of the oligo (M)

[Na+] = Concentration of monovalent ions (M)

Breslauer, K.J.; Frank, R.; Blocker, H. and Marky, L.A. (1986) Proc. Natl. Acad. Sci. USA 83, pp

3746-3750.

The Oligonucleotide T

calculator can be found in the Settings module where the user can input the

oligonucleotide sequence, its concentration in the reaction and the salt concentration of the reaction

mix.

1 2 ... 19 20 21 22 23 24 25 26 27 28 29 ... 68 69

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Techne Prime Elite Manuel d'utilisateur Page 24