I was wondering if I can use a formula for my ISSR primers to calculate the annealing temperature and melting temperature.

Alternatively I would like to know if is possible to calculate with any existing programme online.

Primer 1 CACACACACACACACAG

Primer 2 GAGAGAGAGAGAGAGAC

Primer 3 CACACACACACACACAT

Primer 4 CTCTCTCTCTCTCTCTRG

I am asking this because I normally use the DreamTaq Kit and with the calculator (on Thermo Scientific) I always find some slight differences compared to the Platinum and Phusion protocol regarding the annealing and melting temperature.

The primers have a reference temperature of:

Annealing °C

Primer 1 47

Primer 2 53

Primer 3 47

Primer 4 48

However, I would like to calculate it if possible.

Thanks in advance for your help.

Best regards

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