Abstract
PCR is used to amplify regions to be interrogated for the presence of mutations (SNP and small indel polymorphisms). While PCR is a common practice and many protocols exist, reaction conditions are provided here that are optimized for TILLING and Ecotilling assays utilizing native agarose gel electrophoresis.
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5.1 Materials
Consumables and equipment for PCR amplification are listed in Table 5.1.
5.2 Methods
-
1.
Prepare a PCR master mix on ice by combining:
H2O
82.5 μl
10× Ex Taq buffer
15 μl
2.5Â 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.
Mix the PCR master mix by pipetting it up and down ten times followed by pulse centrifugation.
-
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.
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.
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).
References
Cooper JL, Till BJ, Laport RG, Darlow MC, Kleaffner JM et al (2008) TILLING to detect induced mutations in soybean. BMC Plant Biol 8:9
Rozen S, Skaletsky H (2000) Primer3 on the WWW for general users and for biologist programmers. In: Krawetz S, Misener S (eds) Methods in molecular biology. Humana Press, Totowa, NJ, pp 365–386
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Till, B.J., Jankowicz-Cieslak, J., Huynh, O.A., Beshir, M.M., Laport, R.G., Hofinger, B.J. (2015). PCR Amplification for Low-Cost Mutation Discovery. In: Low-Cost Methods for Molecular Characterization of Mutant Plants. Springer, Cham. https://doi.org/10.1007/978-3-319-16259-1_5
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DOI: https://doi.org/10.1007/978-3-319-16259-1_5
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