Plasmid dna miniprep kit

Genomic DNA was extracted from cells using the PureLink Genomic DNA Extraction Mini Kit (Invitrogen) according to the manufacturer’s instructions. Five-hundred nanogram of purified genomic DNA was then bisulfite converted using the EpiJET Bisulfite Conversion Kit (Thermo Scientific) according to the manufacturer’s instructions. The rDNA locus was amplified from the bisulfite-converted DNA using primers specific to bisulfite-converted sequences in the human rDNA locus^{36 (link)} (GenBank accession code U13369). Primer sequences are as follows: 5′-GTT TTG GGG TTG ATT AGA-3′ and 5′-AAA ACC CAA CCT CTC C-3′. Amplified DNA was cloned into the pCR4-TOPO vector. Individual colonies were isolated and plasmid DNA was purified using a Plasmid DNA Miniprep Kit (Qiagen). Purified DNA was subjected to Sanger sequencing. Clones exhibiting incomplete cytosine conversion were discarded; completely converted clones were analyzed to determine the proportion of methylated to unmethylated CpGs.

The homology of the targeted gene and protein sequences were searched in the Blast program and resources at NCBI¹. Analyses of protein subcellular localization were predicted by WoLF PSORT². Fungal genomic DNA and total RNA extraction were extracted from the mycelia grown in PDB medium for 36 h at 28°C. Fungal genomic DNA extraction was carried out using CTAB as previously described, and total RNA extraction was performed using Trizol (Invitrogen). Plasmid DNA was purified using the Plasmid DNA Miniprep Kit (Qiagen).

Full-length N and S1 proteins of SARS-CoV-2 were synthesized according to the published sequence (General Biosystems (Anhui) Co. Ltd., Anhui, China). The full-length N gene was ligated into the pET-28a vector, which was used to transform DH5α E. coli cells. Colonies were selected on Luria Bertani (LB) agar plates containing 30 µg/mL kanamycin. Recombinant plasmids were extracted using a Plasmid DNA Miniprep Kit (Qiagen, Hilden, Germany), and analyzed for the presence of the N gene by sequencing and the use of BamH I and Xho I restriction enzymes. The recombinant plasmid expressing the S1 gene was acquired and analyzed by the same procedure, except that the pET-30a vector and BamH I and Apa I restriction enzymes were used. To express the N protein and S1 protein, recombinant plasmids encoding the N protein (pET-28a-SARS-CoV-2-N) and S1 protein (pET-30a-SARS-CoV-2-S1) were transformed into E. coli BL21 (DE3) cells. Transformants were selected on kanamycin (30 µg/mL) plates. BL21 (DE3) cells containing recombinant plasmids were cultivated in 5 mL LB broth and the protein expression was induced by the addition of 1 mM IPTG, followed by shaking for 3 h at 37 °C. The bacterial pellet was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) to analyze expression of the recombinant protein.

Using interspecies genetic synteny and sequence homology, PCR primers were designed to amplify the NGN3 gene from pig genomic DNA (gDNA). Using Phusion DNA polymerase (NEB, Ipswich, MA), primers NGN3h-F, 5′-GCCTGCAGCTCAGCTGAACTT-3′, and NGN3c-R, 5′-AGCCAGAGGCAGGAGGAACAA-3′, were used to amplify a 1024 bp product by PCR using gDNA isolated from a mixed breed pig (Poland China/Large White/Landrace). The PCR amplicon was then A-tailed with 2× OneTaq master mix (New England BioLabs), ligated into the TA cloning vector pCR2.1 (Invitrogen, Carlsbad, California), and transformed into competent NEB5α E. coli (New England BioLabs). Plasmids were isolated from transformants using Qiagen Plasmid DNA miniprep kit (Qiagen, Venlo, The Netherlands), and sent to Macrogen USA (Rockville, MD) for sequencing with M13F and M13R primers. Nucleotide BLAST (ncbi.nlm.nih.gov) analysis of the sequencing data confirmed the pig NGN3 gene. Amplicons from twelve independent PCR reactions were cloned into the pCR2.1 vector and sequenced. Sequencing reads for the NGN3 CDS were assembled using the Staden package, version 1.7. A 645 bp fragment corresponding to a single exon was deposited into NCBI Genbank; accession KP796255.

Purified cellular DNA was bisulfite-treated using the EZ DNA Methylation-Lightning Kit (Zymoresearch, Irvine, USA) according to the manufacture’s protocol. To amplify the Rasal1 and Kl promoter fragments, a touchdown PCR program was performed using Taq DNA Polymerase (Sigma-Aldrich, St. Louis, USA). The first round of PCR consisted of the following cycling conditions: 94 °C for 2 min, 6 cycles consisting of 30 s at 94 °C, 30 s at 60–55 °C (reduce 1 °C after each cycle), and 30 s at 72 °C. The second round of PCR consisted of the following cycling conditions: 32 cycles consisting of 30 s at 94 °C, 30 s at 55 °C, and 30 s at 72 °C. The final elongation consisted of 72 °C for 6 min. The sequences of the PCR primers are listed in Supplementary Table 5. The PCR products were purified using the QIAEX II Gel Extraction Kit (Qiagen, Hilden, Germany), cloned into the pGEM-T Vector (Promega, Wisconsin, United States) and transformed into Top10 Competent E.coli Cells (Life Technologies, Carlsbad, USA). The plasmid DNA was then purified with DNA Plasmid Miniprep Kit (Qiagen, Hilden, Germany) and sequenced (Seqlab, Göttingen, Germany).