5 min ta blunt zero cloning kit

Total RNA was extracted from various rice tissues, including root, stem, leaf, sheath and spikelets with anthers at different developing stages, using Plant Total RNA Isolation Kit (Sangon Biotech, Shanghai, China) as described by the manufacturer. The first strand cDNA was synthesized using NovoScript^® Reverse Transcriptase (Novoprotein, Shanghai, China) as described by the manufacturer.
RT-PCR analysis were performed in a regular 28 cycles of three steps PCR program using Biometra TOne thermal cycler (Analytik-Jena, Jena, Germany) with the corresponding primer sets (Table S1) and 2×TSINGKE Master Mix (Tsingke, Beijing, China). The products amplified by RT-PCR with primer Set-FL (Table S5) were further subcloned using 5min TA/Blunt-Zero Cloning Kit (Vazyme, Nanjing, China). More than 200 clones of each plant with different genotype were sequenced for calculating the ratio of each transcript variant. qRT-PCR were performed using qTOWER3G machine (Analytik-Jena, Jena, Germany) with the corresponding primer sets (Table S5) and AceQ Universal SYBR qPCR Master Mix (Vazyme, Nanjing, China). The expression level of Ubiquitin (Os03g13170) was used as the internal control for normalization of mRNA expression ratio.

Total RNA was extracted from a living earthworm using the Mollusc RNA kit R6875 purchased from OMEGA Bio-tec, America. Then, reverse transcriptase-polymerase chain reaction (RT-PCR) was conducted according to the protocol of the HiScript III 1st Strand cDNA Synthesis kit (Vazyme, Nanjing, China) with the oligo (dT)₂₀ primer. Upon the nucleotide sequence of the transcriptome of P. vulgaris, six pairs of primers (Table 6) were designed to be used in PCR amplification with cDNA by the Phanta Max Super-Fidelity DNA Polymerase kit (Vazyme, Nanjing, China). PCR was performed with 3 µL cDNA template, 1 µL DNA polymerase, 1 µL dNTP mix, and 2 µL of each primer in a 50 µL reaction mixture. Additionally, the PCR protocol was as follows: a pre-denaturation for 3 min at 95 °C was followed by 35 cycles of 15 s at 95 °C, 15 s at the right temperature (Tm), and 1 min at 72 °C. The PCR product was purified by the Gel/PCR extraction kit (Biomiga, San Diego, CA, USA) and subcloned into a pCE2 TA/Blunt-Zero vector at 37 °C using the 5 min TA/Blunt-Zero Cloning kit (Vazyme, Nanjing, China). The recombinant plasmids were transformed into E. coli DH5α and sequenced. The sequencing results were compared with the sequences in the transcriptome to further verify the validity of the primary sequences of the purified proteins.

A HiScript-TS 5′/3′ RACE Kit (#RA101-01; Vazyme, Nangjing, China) was used in accordance with the manufacturer’s instructions to perform 5′-RACE, and 3′-RACE of RP11-296E3.2. A 5 min TA/Blunt-Zero Cloning Kit (#C601; Vazyme) was used to generate transformants and the sequences amplified by 5′-RACE, and 3′-RACE were confirmed by sequencing. Additional file 6: Table S1, a list of the gene-specific primers used for RACE.

To detect the mutations that occurred at the target site, genomic DNA was extracted from all T₀ plants and T₁ plants using the CTAB method. PCR for target site amplification was performed using KOD-One (TOYOBO) and the primers listed in Table S2. To determine the mutations introduced by the CRISPR/Cas9 system, the PCR products amplified from different transgenic plants were cloned with the 5min TA/Blunt-Zero Cloning Kit (Vazyme Biotech). The positive colonies were sequenced, and sequences were aligned with WT genome sequences.