PCR Primer Design: How to Pick the Best PCR Primers for Your ExperimentChoosing the right oligonucleotide primers can make the difference between a successful PCR* experiment and a failed one. Here are some tips to help optimize the output and specificity of your experiment. 1. Design PCR primers that are 18-30 oligo nucleotides long. 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 the GC content of each primer in the range of 40-60% for optimum PCR efficiency. 3. Aim for a uniform distribution of G and C nucleotides, as clusters of G's or C's can cause non-specific priming. 4. Make the 3' end slightly AT rich to avoid mispriming. 5. Avoid long runs of the same nucleotide. 6. Keep the melting temperature (Tm) of the primers used within 5°C of each other. Use this formula to calculate Tm: Tm = 4(G + C) + 2(A + T)°C 7. Aim for a Tm between 65 and 70°C for each primer over the region of hybridization. 8. Use an annealing temperature (Ta) of 5 to 15°C lower than the Tm. 9. 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. 10. If you are including a restriction site in the primer, make sure too add 8-10 random nucleotides between the end of the PCR fragment and the restriction site to give the restriction enzyme room to sit on the DNA (e.g. CATGTAGC) These tips should give you a good head start. Redasoft Visual Cloning is another useful tool, integrating your plasmid sequences with the Primer3 PCR primer design functionality. Visual Cloning makes it easy to design optimal primers for your experiment in just a few clicks. Download a free trial here (http://www.redasoft.com). *PCR = Polymerase Chain Reaction References: http://openwetware.org/wiki/Cloning_Checklist http://www.mcb.uct.ac.za//pcroptim.htm http://rothlab.ucdavis.edu/protocols/PrimerDesign.html http://www.protocol-online.org/prot/Molecular_Biology/PCR/PCR_Primer/ http://www.biochem.ucl.ac.uk/bsm/nmr/protocols/protocols/oligo.html |
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