To begin, navigate to the NCBI website to retrieve the required gene from the Japonica Rice variety. Download and access the reference genome within SNAP gene software. Analyze the insertions or deletions and single nucleotide polymorphisms within the exon sequence of O-S-S-B-E 2B from the X134 variety, Comparing it to the reference genome.
using the NCBI primer blasts tool, design primers, flanking the exon regions. To validate the exon sequence of the O-S-S-B-E 2B gene in X134 by Sanger sequencing, prepare the reaction and run the PCR program with the appropriate settings. Design the single guide RNA, or S-G-R-N-A on the O-S-S-B-E 2B exon sequence of X134, adhering to the protocol, and ensure that the proto spacer adjacent motif sequence is TTN.
Add a CAC OLIGOS at the five prime end of the forward primer and GGCC OLIGOS at the five prime end of the reverse primer. After dissolving the primers, mix one microliter of each primer with eight microliters of anneal buffer. Place the mix in the PCR machine and run the annealing program.
Assemble the S-G-R-N-A into the genome editing vector and run the PCR assembly program. Transform the resulting vector into escherichia coli cells, and perform PCR amplification on individual colonies to screen for successful insertions for confirming successful cloning using Sanger sequencing.
