Techne TC-PLUS User Manual
Page 24
22
OLIGONUCLEOTIDE T
M
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
(T
m
) of the primers.
The T
m
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
m
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
m
may result in
reduced priming or prevent priming altogether.
The TC-PLUS uses the Nearest-Neighbour method for calculating the T
m
of an oligonucleotide
1
.
The T
m
is calculated using the following equation:
T
m
=
{(∆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
-1
mol
-1
)
R
=
The Gas Constant (1.99 cal K
-1
mol
-1
)
C
=
Concentration of the oligo (M)
[Na+] =
Concentration of monovalent ions (M)
1
Breslauer, K.J.; Frank, R.; Blocker, H. and Marky, L.A. (1986) Proc. Natl. Acad. Sci. USA 83, pp
3746-3750.
The Oligonucleotide T
m
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.