Techne TC-PLUS 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 TC-PLUS uses the Nearest-Neighbour method for calculating the T

of an oligonucleotide

The T

is calculated using the following equation:

{(∆Hº x 1000)/(A + ∆Sº + R ln(C/4))} - 273.15 + 16.6 x log[Na

]

Where:

∆Hº

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

A = Helix initiation correction constant

∆Sº

= 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.

TEXT ENTRY

When giving a name to a program or stage, or when entering a keyword or a password, a text entry

screen will be opened.

Touch individual characters to create the name.

• For words with all upper case letters touch CAPS.

• Touch SHIFT if only the first letter is required in

upper case.

• For numbers touch 123.

• For commonly used symbols touch SYM.

• Touch the space bar (not labelled) for spaces

between characters.

• To clear the typed name, touch CLR.

• To delete the last character touch DEL.

Once the name is complete, touch OK.

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Techne TC-PLUS Manuel d'utilisateur Page 24

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