Pmd18 t vector system

Genomic DNA from CRC cell lines, CRCs and adjacent NCTs was bisulfite-modified as previously described [39 (link)]. The Bisulfite-treated DNA was amplified using bisulfite-sequencing PCR (BSP) primers located in the promoter region of miR-638. The purified BSP products were directly sequenced and the methylation status of each CpG site was determined as described previously [40 (link)]. The PCR products were cloned using the pMD18-T Vector System (Takara, Japan). Plasmids from single colonies were purified and sequenced. To perform the methylation-specific PCR (MSP) analysis, the bisulfite-treated DNA from the CRC clinical samples was subjected to PCR, and the PCR products were analyzed by 2% agarose gel electrophoresis. The BSP and MSP primers were designed using MethPrimer (Supplementary Table S3) [41 (link)]. HCT-116 or LoVo cells were plated in a 25-cm² flask. Twenty-four hours later, cells were treated with 10 μM demethylating agent, 5-aza-dC (Sigma, USA); the medium was changed at 72 h after treatment. RNA was extracted from the treated cells using the TRIzol reagent and was subjected to a qRT-PCR analysis for miR-638.

The primer sequences of E. coli and 16S were shown in Table S6. PCR products were ligated to the pMD18-T vector system (Takara, Japan), which was subsequently transformed into E. coli strain DH5α. Plasmid DNA carrying the insert was extracted and used as the template for DNA sequencing. Ten-fold serial dilutions of plasmid pMD18-T from 10^-3 to 10^-8 or 10^-3 to 10^-6 were performed to generate standard curve for absolute quantification. Microbial DNA was extracted from the spleen, liver, and colon using the EZNA ^TM Soil DNA kit (Omega Bio-Tek Inc., GA, USA) according to the manufacturer’s protocol. qPCR was performed to quantify E. coli on an ABI 7300 real-time PCR system (Applied Biosystems, USA). The 20-μL PCR reaction mixture contained 10 μL ChamQ SYBR Color qPCR Master Mix (2×), 0.8 μL of each primer, 0.4 μL ROX Reference Dye 1 (50×), and 2 μL of the template DNA. The cycling conditions were as follows: 95°C for 3 min, followed by 40 cycles at 95°C for 5 s, 58°C for 30 s, and 72°C for 1 min.

The standard strain DNAs listed in Table 1 were used to construct the reference plasmids. Plasmids containing the target genes were generated by cloning the PCR products with the pMD18™-T vector system (TaKaRa, Shiga, Japan). All plasmids were defined by sequencing. Plasmid extracts were diluted in ddH₂O to 10⁶ copies/μL in a tenfold dilution series for use in microarray optimization.

Archaeal 16S rRNA genes were amplified from soil community DNA using the primers 21f (5′ TTCCGGTTGATCCYGCCGGA) and 958r (5′ YCCGGCGTTGAMTCCAATT); PCR reactions volume was the same with amplification of full length 16S rDNA of archaea, and PCR reactions were carried out according to DeLong [27 (link)] with modifications as follows: 1 cycle of 95°C for 1 min, 30 cycles of 94°C for 1 min, 50°C for 1 min, 72°C for 2 min, and a final extension at 72°C for 10 min. The PCR products were purified as described above and cloned following the manufacturer's instructions using the pMD18-T vector system (TaKaRa) together with competent E. coli JM109 cells. Randomly selected clones were sequenced.

The total genomic DNA of sediment samples was extracted from 0.5 g (fresh weight) of the sediment sample with the Fast DNA Spin kit for sediments (Qbiogene, Irvine, CA) according to the manufacturer's instructions. After extraction, the DNA samples were immediately frozen at −80°C for further analysis.
The template DNA isolated from the subsamples of each location was pooled so that each subsample was equally represented. The pooled DNA (20 ng for each sample) was analyzed using PCR (predenaturation step of 5 min at 95°C followed by 30 cycles of 1 min at 94°C, 30 s at 53°C and 2 min at 72°C, followed by a final elongation step of 72°C for 15 min), with the bacteria-specific primers 27F (5′-AGA GTT TGA TCM TGG CTC AG-3′) and 1492R (5′-TAC GGY TAC CTT GTT ACG ACT T-3′) (Lane, 1991 ; Meng et al., 2012 (link)). The PCR products were purified using the QIAquick PCR Purification Kit (Qiagen, Germany) and quantified using Nano Drop ND-3000 (Nano-Drop Technologies). The PCR products were subsequently cloned into the pMD18-T vector system (TaKaRa, Japan) and transformed into Escherichia coli Top 10. A total of 2520 recombinant clones were individually chosen from the 30 clone libraries, and partial 16S rRNA gene sequences were determined using a BigDye Terminator V3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA) and an ABI3730 PRISM Genetic Analyzer (Applied Biosystems).

Five microgram genomic DNA of human SF was denatured by 2 M NaOH for 15 min at 50 °C. 2% low-melting agarose were subsequently added to the DNA solution, and agarose beads were formed after pipetting 15-μl DNA/agarose mixture into cold mineral oil. The DNA/agarose beads were treated by freshly prepared hydroxyquinone (10 mM; Sigma) and sodium bisulfite (40.5%, pH 5; Sigma) at 50 °C for 16 h under mineral oil. The reaction was stopped by 0.3 M NaOH for 10 min at room temperature.
The PCR amplifications were performed in 25 μl reactions containing one agarose/DNA bead. The following primer sequences were used: Region 1: 5′-TTTTTATATTAAAGAATTTT-3′ (forward), 5′-TTTATTTATTAAATATGGTGT-3′ (reverse); Region 2: 5′-TAGGTGAAGAAAGTGGTAGA-3′ (forward), 5′-GATTAGATTAATAGGTTAGAA-3′ (reverse) and Region 3: 5′-TTTTTAGTTTTGGAATTGTT-3′ (forward), 5′-AGGTAATATTAGGAGTAGTTTT-3′ (reverse). The PCR products were separated by agarose gel, purified and cloned into the pMD18-T vector system (Takara). Fifteen clones of each sample were sequenced, and the sense strands were used to evaluate CpG site methylation status.

The PCR amplicons were cloned into a plasmid using the PMD18-T Vector system (TaKaRa), and the resulting clones were transformed into Escherichia coli DH5α competent cells (Tiangen, China). The cells were cultured overnight at 37°C on LB media containing ampicillin. One milliliter of this liquid was sent to Shanghai Biological Engineering Company for sequencing. The results were compared with the nucleotide databases in the NCBI GenBank using the BLAST tool.