Since PCR primer design is one of the most widely used features of DNASIS SmartNote, we did some research and put together a list of the top 10 tips for designing PCR primers that work. When designing oligonucleotide primers for PCR, it is helpful to keep some considerations in mind to optimize the output and specificity of your experiment. Here are some tips gathered from experts to get you started:
1. Design your PCR primers to be 18-30 oligo nucleotides 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.
2. Make sure the melting temperature (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
3. Aim for a Tm between 65 and 70°C for each primer over the region of hybridization
4. Use an annealing temperature (Ta) of 10 to 15°C lower than the Tm.
5. The GC content of each primer should be in the range of 40-60% for optimum PCR efficiency.
6. Try to have uniform distribution of G and C nucleotides, as clusters of G’s or C’s can cause non-specific priming.
7. Avoid long runs of the same nucleotide.
8. 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.
9. If you can, make the 3′ end terminate in C or A, as the 3′ is the end which extends and neither the C or A nucleotide wobbles. This will increases the specificity.
10. You can avoid mispriming by making the 3′ end slightly AT rich.
11. Use the right software. OK, so it’s 11 tips. 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. DNASIS SmartNote includes several primer design tools and is also a lab notebook that automatically keeps a record of your analysis results. You should definitely give it a try. Click here to sign up a free account.
If you prefer traditional desktop software, take a look at DNASIS Max instead.