Primer design helper

Warning: This is a very basic tool and should not be seen as a replacement for your brain. This is in no way a complete tool for primer design and shouldn't be trusted! Always check the results yourself. The Tm calculation is based on Belsauer et al. 1986 (with some small modifications, inspired by http://www.basic.northwestern.edu/biotools/oligocalc.html) and has as a goal to match the results found by the NEB Phusion HF-PCR reference (this means that normally the results are the same, but this isn't guaranteed, although the Tm should at least be in the correct range). The tail is ignored in the Tm calculation. Also take a look at iGEM's primer design page.

Known limitations are: only A, C, G and T are accepted as input, the concentration of salts(30mM of Na+), and the primer concentration (500nM) can't be set.

Paste the sequence you want to PCR out in the following textarea:

Give some more info about what you want to do:

Primer type	Standard PCR primer (extract the whole sequence) Add restriction sites on both ends Add "known" tails on both ends Add custom tails on both ends
Restriction sites	Random bases: Primer 1 RS: 5' 3' Primer 2 RS: 5' 3'
Tails	Primer 1 tail: 5'3' Primer 2 tail: 5'3'
Tail
Goal Tm	°C

Result:

Note: the annealing temperatures are indicative and should always be checked (and corrected).
Primer 1 (NaN °C, NaNbp): 5' - No data → 3'
Primer 2 (NaN °C, NaNbp): 5' - No data → 3'
Annealing temp: NaN °C

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