I would like to know how to calculate the Tm of my probe and thus find out the exact and perfect hybridization and washing temperature if I am using formamide in my hybridization buffer. I have gone through several websites but none is giving the exact description. I am using non radioactive method for the process Can anyone help?
Use the following equation to calculate the Tm based on the length of your probe. Tm = 81.5 + 16.6 (log10 [Na+]) + 0.41 [(G + C)/n)(100)] – 600/n where n = number of nucleotides and the concentration of Na+ is 1.0 M or less. For DNA molecules longer than 200 nucleotides, perform the hybridization at 15–25°C below the calculated melting temperature (Tm) of a perfect hybrid.
You can use probe primer Tm for hybridization temperature and for primary stingency washes
As to Palle it said use Tm primer, but before you can try a first hybridization with 55°C, because this Tm is appropriate for almost probe assays.
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