Sigma dna extraction kit

Total DNA of each variety was extracted from leaves of two-week-old germinated seedling using Mutou et al.'s protocol [42 (link)] and Sigma DNA extraction kit. DNA quality and quantity were analysed using NanoDrop spectrophotometer. The integrity of DNA samples was determined using 0.8% agarose gel. The DNA samples were sequenced using Illumina HiSeq 4000 sequencing (Illumina, Inc., San Diego, CA, USA). Standard Illumina protocol was used for the sequencing process.

K14E6hK14E7h mice were obtained by crossing K14E6H females with K14E7h heterozygous females. The mice are kept on a pure FVB genetic background. The presence of E6 and E7 transgenes was confirmed through PCR genotyping, using DNA extracted from mouse tail with Sigma DNA extraction Kit (Sigma-Aldrich, cat. # G1N10). E7 transgene was detected using (Oligo2/ E6TTL) primers, and E6 was detected using (K14709–4/E7TTL) primers as previously described [28 (link)]. PCR reactions were carried out using KAPATaq (Kappa Biosystems, cat. # KK1015).

Genomic DNA of the above-mentioned 227 (110 + 117) F₂- and BC₁-derived inbred lines and their seven parents (B. napus A04-73NA and six B. oleracea cultivars/lines) was extracted using SIGMA DNA extraction kit (Sigma-Aldrich, St. Louis, MO, USA) following manufacture's instruction. A total of 95 polymorphic SSR markers (Nikzad et al., 2019 (link)) from nine C-genome linkage groups (LG) were selected from 418 markers for genotyping the populations. Polymerase chain reactions (PCR) for amplification of the genomic DNA was performed in a total volume of 15.5 µl, which included 20 ng genomic DNA, 5× PCR reaction buffer, 25 mM MgCl₂, 0.6 unit Taq DNA polymerase (Promega Corporation, Madison, WI), 10 mM each dNTP (Invitrogene Life Technologies Inc., Burlington, ON), 5 µM of each forward and reverse primer, and 5 µM tag F (fluorescent dyes FAM, VIC, NED, and PET). PCR was carried out in a GeneAmp PCR System 9700 thermal cycler (Applied Biosystems, Foster City, CA) with the following program: 1 cycle of 5 min at 95°C for initial denaturation, 35 cycles where each cycle consisted of 1 min at 95°C for denaturation, 1 min at 58°C for annealing and 1.5 min at 72°C for extension, and the final extension time was 15 min at 72°C. Size-based separation of the amplified DNA fragments was done using a capillary electrophoresis AB Genetic Analyzer No. 3730 (Applied Biosystems, Foster City, CA).