The annealing stage is the most important and is a variable step in the PCR process.
Hi, is there no supervisior who can help you? You have no literature to read about the topic?
http://www.news-medical.net/health/Oligonucleotide-What-is-an-Oligonucleotide.aspx
http://users.ugent.be/~avierstr/principles/pcr.html
http://www.nfstc.org/pdi/Subject04/pdi_s04_m01_02_c.htm
http://www6.appliedbiosystems.com/support/techtools/calc/
Cheers, Nadine
You need to determine which annealing temperature is suitable for the primers that you have, not the other way around as its stated in your question. Basically, primers are pieces of 'code' that will bind to the complimentary nucleotides of the region of the gene you are interested in amplifying using PCR.
First, you need to design your primers for your specific gene (https://bioweb.uwlax.edu/GenWeb/Molecular/seq_anal/primer_design/primer_design.htm). Then once you have received them, you have to figure out its annealing temperatures and cycle numbers for each primer (depending on where you order your primers from, the company usually gives a recommended temperature, which always has to be tested by you, but at least you have a starting point). Otherwise you can use this: https://www.neb.com/tools-and-resources/interactive-tools/tm-calculator.
Now for the cycle number, I would usually do cycles 20, 22, 24, 26, 28, 30 to be able to calculate a 'linear phase' (http://www.sourcemolecular.com/images/amplification_plot_new.gif) where you can then determine the optimal cycle number.
Hope that makes sense and I explained it right! Its been a while since my last PCR!
- Badges
- Science method