Primer designing for PCR


Design your PCR primers to be 18-30 oligonucleotides in length. The longer end of this range allows higher specificity and gives you space to add restriction enzyme sites to the primer end for cloning. Make sure the melting temperatures (Tm) of the primers used are not more than 5°C different from each other. You can calculate Tm with this formula: Tm = 4(G + C) + 2(A + T)°C. Aim for a Tm between 52 and 58°C for each primer over the region of hybridization. Use an annealing temperature (Ta) of 3-5°C lower than the Tm. The GC content of each primer should be in the range of 40-60% for optimum PCR efficiency. Try to have uniform distribution of G and C nucleotides, as clusters of G’s or C’s can cause non-specific priming. Avoid long runs of the same nucleotide. Check that primers are not self-complementary or complementary to the other primer in the reaction mixture, as this will encourage formation of hairpins and primer dimers and will compete with the template for the use of primer and reagent. If you can, make the 3′end terminate in C or A, as the 3′is the end which extends and neither the C nor A nucleotide wobbles. This will increase the specificity. You can avoid mispriming by making the 3′end slightly AT rich. Use the right software. Using the right software is a great way to automate these steps and minimize errors, especially when you have to design primers for many sequences.

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