Sureplant dna kit

Total RNA was extracted from chrysanthemum using TRIzol Reagent (Invitrogen, 15596-026). cDNA was synthesized using SuperScript III reverse transcriptase (Invitrogen, 18080-044). Degenerate primers were designed based on a sequence alignment of CCD7 from different species (Supplementary Table S3). A fragment of the partial DgCCD7 gene was obtained, which was extended using Rapid Amplification of cDNA Ends (RACE) PCR. Amplified fragments were cloned into the pMD18-T vector (Takara, D101A) and sequenced. Genomic DNA was isolated from young tissue using the SurePlant DNA Kit (Cwbio, CW0555). Amplified products were used to determine genomic clones and intron positions in DgCCD7 genes.

Global 5mC levels in tomato genomic DNA was determined as previously described with minor modifications [68 (link)]. In brief, DNA was extracted from the pericarp tissues using Sureplant DNA kit (Cwbiotech, CW2298), with the disruption of total RNA according to the manufacturer’s protocols. The extracted DNA was detected in 1% agarose gel and quantified by a SimpliNano spectrophotometer (GE Healthcare, 29-0617-11). Then, 100 ng of purified and integrated DNA for each measurement was used to perform 5mC assay by MethylFlash™ methylated DNA quantification kit (Epigentek, P-1034). 5mC levels in different DNA samples were relatively quantified using both the negative control and positive control, which contain 0% 5mC and 50% 5mC, respectively, following the manufacturer’s instructions.

Genomic DNA was extracted from fresh trifoliate leaves of 2-week-old seedlings with a SurePlant DNA kit (CWBIO) and used to amplify indel markers. The primer sequences used to amplify the markers for mapping are listed in Supplementary Table 9. For fine mapping, dCASP markers were developed in the regions of Tof16 based on the resequencing data of the two parents, PI591429 and PI628930. Seven recombinants were identified in the fine-mapping population of Tof16 using seven markers. The flowering time of the progeny of these recombinants was evaluated to delimit the genomic interval containing Tof16.