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5.1 Materials

Consumables and equipment for PCR amplification are listed in Table 5.1.

Table 5.1 Chemicals, enzymes, and equipment for PCR amplification
Full size table

5.2 Methods

  1. 1.

    Prepare a PCR master mix on ice by combining:

    H2O

    82.5 μl

    10× Ex Taq buffer

    15 μl

    2.5 mM dNTP mix

    12 μl

    10 μM L primer

    1.5 μl

    10 μM R primer

    1.5 μl

    TaKaRa HS taq (5 U/μl)

    0.38 μl

  2. 2.

    Mix the PCR master mix by pipetting it up and down ten times followed by pulse centrifugation.

  3. 3.

    Combine 7.5 μl DNA at the appropriate concentration with 22.5 μl of PCR master mix. Mix by pipetting it up and down.

  4. 4.

    Incubate in a thermal cycler using the following parameters:

    95 °C for 2 min; loop 1 for 8 cycles (94 °C for 20 s, 73 °C for 30 s, reduce temperature 1 °C per cycle, ramp to 72 °C at 0.5 °C/s, 72 °C for 1 min); loop 2 for 45 cycles (94 °C for 20 s, 65 °C for 30 s, ramp to 72 °C at 0.5 °C/s, 72 °C for 1 min); 72 °C for 5 min; 99 °C for 10 min; loop 3 for 70 cycles (70 °C for 20 s, reduce temperature 0.3 °C per cycle); hold at 8 °C.

  5. 5.

    OPTIONAL: Check the yield of the PCR product by agarose gel electrophoresis. See Chap. 8 for example data. For the efficient discovery of nucleotide polymorphisms, PCR product yield should be approximately 10 ng/μl or higher in concentration. PCR product should be a single band. Co-amplification of multiple sequences can result in high error rates (Cooper et al. 2008).