I have designed a primer for Bacillus subtilis sfp gene. As I have mentioned the size is 191 bp. however I need some suggestions regarding PCR temperatures and cycle length. As the it is smaller size, should I use only 20 cycles?
UPDATE: I meant 30 cycles. it was a typing mistake.
It depends on how many copies of your target dna there are to start with but I would start with 35 cycles and if there is too much non specific amplification then reduce the cycles but 20 is not enough for normal use. the number of cycles is not related to the length of the amplimer but the really important thing in new PCR reactions is to get the annealing temperature of the primers right. Start at about 5c below the melting temperature and work upwards or run a temperature gradient if your pcr machine can do this
It depends on how many copies of your target dna there are to start with but I would start with 35 cycles and if there is too much non specific amplification then reduce the cycles but 20 is not enough for normal use. the number of cycles is not related to the length of the amplimer but the really important thing in new PCR reactions is to get the annealing temperature of the primers right. Start at about 5c below the melting temperature and work upwards or run a temperature gradient if your pcr machine can do this
I think it’s better you run an insilico pcr with different annealing temperature starting with a temperature of 5 degree celsius below your melting temperature and then continue to increase by 1degree until full optimization Of your cycling condition. This way you will be able to save time and consumables during optimization. In an alternative if you have a gradient pcr machine then you can run a gradient temperature.
i will also suggest you start with 30-35 cycles.
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