Amplifying NaN1793 Mutant Region and Confirming Presence of the Mutation

Using the Primer3 software, primers were created to amplify the region of the NaN1793 mutation. These primers were chosen with respect to the fact that Sanger sequencing can only sequence about 1000 b.p. maximum, and the mutation should not be too close to either primer since the beginning and end of sequencing data is usually less accurate than the middle. The expected size of the amplicon is 874 b.p. In order to confirm that DNA was extracted from the plants and to decide which samples to take for sequencing, gel electrophoresis was run. Two gels were run. One gel contains PCR products in a ¼ dilution in T10E1 (TE) buffer while the other contains PCR products in a ¼ dilution with H2O as a control. The T10E1 diluted DNA was preferentially taken for sequencing because it protects DNA from degradation, so that DNA should be of better quality than the H2O diluted DNA. Because there was no band in the Mutant 1 lane of the TE gel, Mutant 1 was taken from the H2O gel. Mutant 2, Mutant 6, and Mutant 7 were all taken from the TE gel. The DNA was extracted and purified from the gel and was nanodropped to confirm presence of DNA and assess purity. In order to perform Sanger sequencing, a primer is needed. The forward primer is 428 b.p. from the mutation site, while the reverse primer is 447 b.p. from the mutation site. The forward primer was used for sequencing because it is closer to the mutation site.