Nuclean plant genomic dna kit

DNA was extracted from dried specimens with the NuClean Plant Genomic DNA kit (CWBIO, China). Three regions (ITS, LSU and Tef-1α) were generated for the study, which were amplified with primers ITS1/ITS4 (White et al. 1990 (link)), LR0R/LR7 (Hopple and Vilgalys 1999 (link)) and EF983F/EF2218R (Örstadius et al. 2015 (link)), respectively. PCR was performed using a touchdown programme: 5 min at 95 °C; 1 min at 95 °C; 30 s at 65 °C (add -1 °C per cycle); 1 min at 72 °C; 15 cycles; 1 min at 95 °C; 30 s at 50 °C; 1 min at 72 °C; 20 cycles; and 10 min at 72 °C (Yan and Bau 2018 (link)). The DNA sequencing was done by Qing Ke Biotechnology Co. Ltd. (Wuhan City, China).

The gene-edited rice CYP81A6 knockouts by using CRISPR/Cas9 and adenine base editor (ABE) were made in our laboratory. The NuClean Plant Genomic DNA Kit was purchased from CWBIOTECH Co., Ltd. (Jiangsu, China). The PCR reagent was purchased from Takara Biology Co., Ltd. (Dalian, China). The DNA Constant Temperature Rapid Amplification Kit (Basic) was purchased from Weifang Anpu Future Biotechnology Co., Ltd. (Weifang, China). The HiScribe T7 High Yield RNA Synthesis Kit and EnGen Lba Cas12a (Cpf1) were purchased from New England Biolabs (Ipswich, MA UK). RNA Clean & Concentrator-25 was purchased from Zymo Research (California, USA). All of the primers were synthesized by Sangon Biotech (Shanghai, China). The polyethylene filter membrane (aperture, 10 μm), silica gel adsorption membrane (aperture, 1 μm), and screw column were purchased from Hangzhou Laifeng Biotechnology Co., Ltd. (Hangzhou, China).

Genomic DNA was extracted by using NuClean Plant Genomic DNA Kit (CWBio, Beijing, China). PCR reactions were performed by specific primers using standard procedures. The PCR products were sequenced by specific primers. If the sequencing results contained mutations, the PCR band was extracted from the agarose gel, then purified and cloned into pEASY T5-ZERO vector (TransGen Biotech, Beijing, China). Five individual E. coli colonies were selected for plasmid preparation (TIANprep Mini Plasmid Kit, TIANGEN, Beijing, China) and sequencing by Sanger method.

Isolates were cultured on SDA for 72 h prior to DNA extraction. Genomic DNA was extracted with the NuClean Plant Genomic DNA Kit (CWBIO, Beijing, China). Based on the ISHAM consensus MLST scheme, seven loci including capsule polysaccharide (CAP59), glycerol 3-phosphate dehydrogenase, (GPD1), laccase (LAC1), the intergenic spacer (IGS1) region, phospholipase B1 (PLB1), superoxide dismutase (SOD1), and orotidine monophosphate pyrophosphorylase (URA5) genes were amplified and sequenced[5 (link)]. The alleles were analyzed, and the STs were determined based on the MLST database (http://mlst.mycologylab.org). The sequences of the seven MLST loci have been deposited in the MLST database and GenBank.
Phylogenetic analysis of the C. gattii species complex was performed based on the alignment of seven concatenated nucleotide sequences (CAP59, GPD1, LAC1, IGS1, PLB1, SOD1 and URA5)[21 (link)]. The genetic relationships among these Chinese clinical strains and strains in different countries[21 (link)–23 (link)] were investigated by MEGA7[24 (link)]; a phylogenetic tree was generated using the maximum likelihood method with a bootstrap analysis using 1000 replicates[24 (link)–26 (link)]. Principal component analysis (PCA) was performed with the Adegenet 2.1.1 package for software R (version 3.4.4) to explore the genetic relationships and geographic patterns of the strains[27 (link)].

DNA was extracted from freesia flowers using NuClean Plant Genomic DNA Kit (CWBIO) according to the manufacturer’s instruction. Promoters of FhDFR1/FhDFR2 and FhDFR3 were cloned using Genome Walking Kit (TaKaRa) following the instructions. The -1466 bp of FhDFR1/FhDFR2 and -1132 bp of FhDFR3 from the initiation condon “ATG” were amplified as promoters and cloned into Pst I and Sac I digested AtDFR-pro:GUS construct to generate FhDFR1/FhDFR2-pro:GUS and FhDFR3-pro:GUS, respectively. All the other constructs used for protoplasts transfection have been described previously (Li et al., 2016 (link)). All the plasmids were prepared using the EndoFree Plasmid Maxi Kit (CWBIO) following the manufacturer’s instructions.

DNA was extracted from dried specimens with the NuClean Plant Genomic DNA kit (NuClean Plant Genomic DNA kit">CWBIO, China). Four regions (ITS, LSU, tef-1α and β-tub) were amplified for the study, which using ITS1/ITS4 (White et al. 1990 (link)), LR0R/LR7 (Hopple and Vilgalys 1999 (link)), EF983F/EF2218R (Örstadius et al. 2015 (link)), and B36f/B12r (Nagy et al. 2011 (link)), respectively. PCR was performed using a touchdown program for all regions as follows: 5 min at 95 °C; 1 min at 95 °C; 30 s at 65 °C (add -1 °C per cycle); and 1 min at 72 °C for a cycle of 15 times; 1 min at 95 °C; 30 s at 50 °C; and 1 min at 72 °C for a cycle of 20 times; and 10 min at 72 °C (Yan and Bau 2018b (link)). DNA sequencing was performed by Qing Ke Biotechnology Co., Ltd. (Wuhan City, China), using primers listed above.