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Conditions for Selecting a Gene-Specific Fragment
Minimum length of the fragment to design primer
If the length of the unqiue fragment selected from is shorter
than this, no primer will be designed.
Conditions for Selecting Primers
Primer Size
Minimum, Optimum, and Maximum lengths (in bases) of a primer oligo.
Min cannot be smaller than 1.
Max cannot be larger than 36.
Primer Tm
Minimum, Optimum, and Maximum melting temperatures (Celsius)
for a primer oligo.
The oligo melting temperature is calculated based on
Rychlik, Spencer and Rhoads, Nucleic Acids Research, vol 18, num
12, pp 6409-6412 and Breslauer, Frank, Bloeker and Marky,
Proc. Natl. Acad. Sci. USA, vol 83, pp 3746-3750.
Maximum Tm Difference
Maximum acceptable (unsigned) difference between the melting
temperatures of the left and right primers.
Primer GC%
Minimum, Optimum, and Maximum percentage of Gs and Cs in any primer.
Max Complementarity
The maximum allowable local alignment score when testing a single
primer for (local) self-complementarity and the maximum allowable
local alignment score when testing for complementarity between
left and right primers.
Max 3' Complementarity
The maximum allowable 3'-anchored global alignment score when
testing a single primer for self-complementarity, and the maximum
allowable 3'-anchored global alignment score when testing for
complementarity between left and right primers.
Max Poly-X
The maximum allowable length of a mononucleotide repeat,
for example AAAAAA.
Salt Concentration
The millimolar concentration of salt (usually KCl) in the PCR.
Primer3 uses this argument to calculate oligo melting
temperatures.
DNA Concentration (Annealing Oligo Concentration)
The nanomolar concentration of annealing oligos in the PCR.
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