Takara minibest agarose gel dna extraction kit

The viral genomic DNA or RNA was extracted using the QlAamp Viral DNA/RNA Mini Kit (QIAGEN, Hilden, Germany). RNAs were reverse-transcribed using the PrimeScript™ RT Reagent Kit (TaKaRa BIO Inc., Dalian, China). Salmonella, Pm, MRSA, HPS, and S. suis were cultured, and the genomic DNA was extracted using TaKaRa MiniBEST Bacteria Genomic DNA Extraction Kit (TaKaRa). All DNA and cDNA were stored at −20°C.
The target genes of the nine porcine viral pathogens were amplified using their specific primer pair. The amplified products were purified using the TaKaRa MiniBEST Agarose Gel DNA Extraction Kit (TaKaRa BIO Inc., Dalian, China). The purified DNA was ligated into the pMD19-T vector, and the ligated constructs were transformed into E. coli DH5a cells cultured in the presence of ampicillin (100 μg/mL). The recombinant plasmid construct was confirmed by DNA sequencing (Life Technologies Inc., Shanghai, China), and the sequence data were analyzed using DNASTAR software and compared with the corresponding sequence data in GenBank.

Proximal promoter regions of SLC2A2 in bats were amplified using two pairs of primers (see Table 4). The following polymerase chain reaction (PCR) protocol was used: pre-denaturation at 95 °C for 5 min, followed by 30 cycles of 30 sec at 94 °C, 30 sec at 45–50 °C and 20 sec at 72 °C, and a final elongation of 10 min at 72 °C. PCR fragments were then separated on 1% agarose gels and purified with a TaKaRa MiniBEST Agarose Gel DNA Extraction Kit (Takara, Japan). Products were then cloned into pGEM-T vectors (Promega, USA), propagated in DH5α or TOP10 competent cells (TIANGEN, China), and sequenced using Big Dye Terminator on an ABI 3730 DNA sequencer (Applied Biosystems, USA). SLC2A2 proximal promoter sequences were amplified twice from one individual of each species for cloning.

The PCR products of the expected size (~800 bp) were purified using the TaKaRa MiniBEST Agarose Gel DNA Extraction Kit (TaKaRa, Japan) and were cloned into pMD19-T Vector (TaKaRa, Japan). The vector was transformed into E. coli strain DH5α, which were spread on agar plates and incubated overnight at 37°C. Inserts from 80 randomly selected clones in each resulting SSU rRNA gene library (4 blocks × 4 growth stages × 2 years × 2 cultivars = 64 libraries) were sequenced using M13-f47 and M13-r48 primers on ABI 3730 genetic analyzer. The obtained sequences were trimmed to remove the vector sequence, and sequence similarities were determined using the Basic Local Alignment Search Tool for nucleotides (BLASTn) sequence similarity search tool provided by GenBank [24 (link)]. Sequences were clustered at 97% sequence similarity with QIIME [25 (link)] and a representative sequence from each OTU was picked for downstream analysis. Taxonomic assignments of OTUs were performed by using QIIME in accordance with SILVA 115 [26 (link)]. Representative sequences of the clones generated in this study were deposited at the National Centre for Biotechnology Information (NCBI) GenBank database under the accession numbers KJ740816 to KJ740970.

PCR products were purified using the TaKaRa Mini BEST Agarose Gel DNA Extraction Kit (Takara, Japan) and sequenced on an ABI-3730 DNA analyzer (Applied Biosystems, Foster City, CA, USA).The sequences were analyzed using BLAST (http://ncbi.nlm.nih.gov/). Phylogenetic trees of bacteria and fungi were separately constructed using the neighbor-joining method (NJ; Saitou & Nei, 1987 (link)) implemented in MEGA 6.0 (LynnonBiosoft, San Ramon, CA, USA). The sequences of bacteria and fungi were submitted to GenBank using SEQUIN (see phylogenetic trees for accession numbers).

The partial HIF-1α for Aurelia sp.1 (ASHIF) nucleic acid sequence was obtained from RNA-seq (PRJNA219043) generated by IOCAS. Degenerate primers (CTF653 F0 and CTF653 R0, Table 1) were designed for confirmation of the partial ASHIF gene sequence. A full-length ASHIF transcript was obtained from the total RNA of Aurelia sp.1 medusa using 5′ and 3′ end RACE. For the 5′ end RACE, the cDNA was synthesized via reverse transcription (RT) using the SMARTer RACE cDNA Amplification Kit (Clontech, Mountain View, CA, U.S.A.), and the PCR amplification was performed with the CTF653 R6 and CTF653 R5 primers (Table 1) using Clontech Advantage PCR Kit (Clontech, Mountain View, CA, U.S.A.). Similarly, the cDNA from the 3′ end RACE was synthesized by RT using the 3′-Full RACE Core Set with PrimeScript RTase (Takara, Dalian, China), and the PCR amplification was performed with the CTF653 F2 and CTF653 F3 primers (Table 1) using TaKaRa LA Taq with GC Buffer (Takara, Dalian, China). All products from the 3′ end and 5′ end RACE PCRs were gel-purified using the TaKaRa MiniBEST Agarose Gel DNA Extraction Kit (Takara, Dalian, China) and cloned into Escherichia coli using a T-Vector pMD (Takara, Dalian, China). Three positive clones were randomly selected for each gene product and sequenced.

MethPrime program was used to predict the potential methylation sites of Serpine1 and design the primers (Serpine1-BSP-F: 5′-GGAATTTGATATTTTTTAGTTTTTATATG-3′ and Serpine1-BSP-R: 5′-CCTACTTCTACCTCCTAAATACTCC-3′) for bisulfite sequencing PCR (BSP). Bisulfite conversion and purification were performed using the EpiTect bisulfite Kit (Qiagen) per the manufacturer’s instructions. Bisulfite-converted DNA was amplified with TaKaRa EpiTaq HS (TaKaRa), and the 139-bp PCR product was purified with the TaKaRa MiniBEST agarose Gel DNA Extraction Kit (TaKaRa). Amplified fragments were inserted into the pMD^™18-T vector (TaKaRa) and transformed into Escherichia coli Trans5α. Plasmid DNA from individual clones was isolated and subjected to Sanger sequencing using M13F primers. The methylation status of each CpG locus was determined by sequence comparison using the QUMA software.