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There are many computer programs available that can be used to design PCR primers, and their use is strongly recommeded, as these programs tend to incorporate several different parameters that the manual design of primers may fail to consider (i.e., Tm, intra- and/or intermolecular complementarity, mispriming libraries, etc.). We have had success using Primer3, which is available for use as an online tool (see http://frodo.wi.mit.edu/primer3/input.htm) as well as being freely available for download. References: Korbie D J, Mattick J S. Touchdown PCR for increased specificity and sensitivity in PCR amplification[J]. Nature Protocols, 2008, 3(9):1452-6. |
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