PCR. Annealing Temperature, Genomic DNA, Conventional PCR, PCR Primer design, DNA Amplification.
There is no general perfect temperature. The annealing temperature will be entirely dependent upon the primers you use and to some degree the template. The elongation temperature will depend upon which enzyme you use (but this is standardized for each enzyme).
Michael J. Benedik can you tell me some specific annealing temperature ? That I can run PCR and try more time.
It is impossible for anyone to give you annealing temperatures without knowing the sequence of your primer and the degree of homology to your template. However there are a number of websites (such as where you ordered your primers from oftentimes) that will have an online calculator and you can input your primer sequence and it will generate your annealing temperatures. But if you have any added bases to your primer like for a restriction site, but sure to remove those and only calculated based on the segment of the primer that is homologous to the template.
You need to know the product size of your primer.
And the TM and Ta of the primer
Then only you can set up up the optimal annealing temperature for your reaction.
A general rule of thumb is use an annealing temperature about 5deg below the TM(degC) of the primers. This presumes the primers have 100% homology over their entire length. So for the primers in the table you added, I would try 59C to start with.
the thermal cycling conditions were as follows: 2 min at 94 °C; 10 cycles of 45 sec at 94 °C, 1 min at 65 °C (annealing temperature was reduced 1 °C after every cycle), and 1 min and 30 sec at 72 °C; 35 cycles of 45 s at 94 °C, 1 min at 55 °C, and 1 min and 30 s at 72 °C; and a final extension step of 5 min at 72 °C. ( source : http://www.scielo.br/pdf/sa/v69n5/a07v69n5.pdf )
Or if you have a pcr with a gradient property, you can find what you are looking for much easier.
Mehmet KOÇ Michael J. Benedik thank you for your valuable suggestion. I already got band at 64'C .
- Badges
- Science method