![]() If you are lucky enough to have access to a thermocycler with a gradient function and enough template to set up the PCR in duplicate or triplicate, I’d suggest trying a range of annealing temperatures and assessing which T a provides the best results. For instance, if your forward primer has a T m of 62☌ and your reverse primer has a T m of 61☌, I suggest starting with a T a of 58☌. I recommend that the annealing temperature is set 3☌ below the lowest melting temperature of your primers. The OligoAnalyzer tool will also check for any secondary structures when designing oligos. This will prevent the oligos themselves from being amplified during the PCR. Double check that any unwanted secondary structures have a melting temperature significantly below your ideal annealing temperature (T a) (i.e., at least 10☌ lower). The melting temperature is affected by the buffer salt concentrations, pH, and primer concentration, but I’ve found this to be of minimal concern. Where A, T, G, and C equal the number of each of those bases in the primer sequence. However, if you’d prefer to do it yourself, here is the equation: The OligoAnalyzer tool on the IDT website is really useful when determining the optimal annealing temperature to set in your PCR cycles. However, I’ve noticed that different companies estimate the T m slightly differently, depending on the calculation they use. The company that you order your oligo primers from will include the melting temperature, or T m, in the documentation they send with the oligos. (Although, the gradient function is pretty handy.) Melting and Annealing Temperatures Here are some tips to get you there faster. But unless you have a never-ending supply of template, polymerase, and a thermocycler with a gradient function-not to mention a hefty dose of time and patience-you probably don’t want to spend the next week finding the perfect conditions for your PCR. So, you’ve designed PCR primers to amplify your sequence of interest, and you’re ready to go. ![]()
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