The NaN1898_Bd1_70884553_Het mutation was located on the Bradi72430 gene as a point mutation converting a guanine to adenine. Through Primer 3, primers for PCR of the mutation site were identified: GGGGTTCTTGTCTGCGCTCTGGT (left) and GCACACGAGAGGAAAACGACCGC (right). The primers were for a product of 928 bases, and the mutation was 134 bases away from the left primer (Figure 1).
DNA was successfully extracted from six mutant plants and two wildtype plants. PCR of this DNA was performed with an annealing temperature of 66°C. The resulting PCR products were visualized via gel electrophoresis. Bands on the gel of PCR products diluted double distilled H2O (Figure 2) were much more prominent, indicating higher DNA content than PCR products diluted with T10E1 (Figure 3). Bands were present in lanes 2-5 and 7-8 of both gels about 3.5 cm from the wells. There were no bands in lanes 6 and 9 of both gels. Bands in lanes 2-5 (mutants 1-4) of gel 2 were the brightest and least smeared overall, so these were used for extraction and purification. Once extracted and purified, the DNA of mutants 1 proved to have the highest DNA content (Table 1). The A260/A280 ratios revealed the DNA of mutants 1 and 4 to be relatively pure, with ratios close to 2.0. DNA from mutants 2 and 3 were more contaminated. These four samples of extracted and purified DNA were sent for Sanger sequencing.
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