Primer concentration and TM value technique
Primer concentration calculation:
Primers are stable at high concentrations. The primers are generally formulated to be 10-50 pmol/ul. In general, it is recommended to set the concentration of the primer to 50 pmol/ul, and the volume of water (microliter) is calculated as follows: V (microliter) = OD number * (multiplication) 33 * (multiplication) * (multiplication) 20000 / (except) the molecular weight of the primer. The molecular weight of the primers can be obtained from the synthesis report. If you need to prepare other concentrations, convert according to the above formula.
Note: 1 OD260 = 33 ug/ml.
Calculation of primer Tm value
Primer design software can give Tm, which is related to primer length, base composition, and the ionic strength of the primers used for buffering.
For primers with a length of 25 mer or less, the Tm is calculated as: Tm = 4 ° C (G + C) + 2 ° C (A + T)
For longer oligonucleotides, the Tm is calculated as:
Tm = 81.5 + 16.6 x Log10[Na+] + 0.41 (%GC) – 600/size
In the formula, Size = the length of the primer.
Definition of Tm: Tm = Temperature at which 50% of a given oligonucleotide is hybridized to its complementary strand. In the absence of destabilizing agents, like formamide or urea, Tm will depend on 3 major parameters: The sequence: a GC-rich sequence The salt concentration: high oligonucleotide concentrations favor hybrid formation, which results in a higher melting temperature. The salt concentration: high ionic strength results in a higher Tm as cations stabilize the DNA duplexes.
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