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7 protocols using sau3ai

1

Histochemical Staining with DAB and DAPI

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Paraformaldehyde (PFA) was purchased from Merck (Darmstadt, Germany), and 3,3'-diaminobenzidine 4HCl (DAB) was purchased from Dojin Chemical Co. (Kumamoto, Japan). Bovine serum albumin (BSA) (essentially fatty acid and globulin-free), Trizma base and Brij-35 were from Sigma Chemical Co. (St. Louis, MO, USA). Biotin-16-dUTP, digoxigenin-11-dUTP and terminal deoxynucleotidyl transferase (TdT) were from Roche Diagnostics (Mannheim, Germany). Dideoxy ATP (ddATP) and dideoxy TTP (ddTTP) were from Jena Bioscience (Jena, Germany). Hpa II, Msp I, Sau3A I and Mbo I were purchased from Takara Bio Inc. (Shiga, Japan). 4',6-Diamidino-2-phenylindole (DAPI) was from DAKO (Glostrup, Denmark). Permount was purchased from Fisher Scientific Inc. (NJ, USA). All other reagents used in this study were from Wako Pure Chemicals (Osaka, Japan) and were of analytical grade.
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2

Functional Screening of Soil Metagenome

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LB agar plate with 100 μg/mL ampicillin and 2.5 mM DET was used for function-driven screening of a soil metagenomic library (1.3 × 105 clones). After the library clones were incubated at 37 ℃ for 24 h, colonies that showed clear halos around them on the agar plate were identified as positive clones and were picked for the next analysis. To further identify the specific hydrolase gene, the plasmids of positive clones were extracted using commercial kits and then partially digested by Sau3AI (Takara, Japan). The resulting 1–5-kb DNA fragments were recovered, subcloned into the pUC118 vector, and transformed into E. coli DH5α. Similarly, the positive subclone was identified according to DET-degrading activity on the LB agar plate and sequenced at Tsingke Biotech Co., Ltd. (Nanjing, China).
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3

Identification of Tetracycline Resistance Gene

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The gene responsible for tetracycline resistance was identified by subcloning. The cosmid DNA from the resistant clone was partially digested with Sau 3AI (Takara Bio, Dalian, China), and DNA fragments of 2–3 kb were then recovered and ligated into Bam HI digested and alkaline phosphatase treated pTG19-T vector (Generay Biotech, Shanghai, China), electroporated into E. coli EPI100-T1R, and plated onto LB screening agar plates for resistant subclones. All screened subclones were verified by restriction digestion and retransformation to confirm the phenotypes. The resulting positive recombinant clones were sequenced by using T7 promoter and M13 primers at first, then by primer-walking sequencing.
Sequence analysis was carried out with the BLASTx program1. Amino acid alignment was performed with the ClustalW2.0 program and phylogenetic analysis (Neighbor-joining tree) was done with MEGA6.0 using neighbor-joining (500 bootstrap replicates).
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4

SNP Genotyping for Chinese Populations

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Six genome-wide CAT-associated SNPs (rs1107877, rs11185115, rs12202737, rs12626864, rs13729, and rs9825563) were experimentally confirmed in Chinese populations. Genomic DNA was extracted from whole blood using the standard phenol/chloroform method. Among them, rs11185115 within NTNG1 was replaced by a nearby SNP, rs1444041 (r2 = 0.954), which was not in the original dataset, owing to a lack of the appropriate SNPstream primers; rs12202737 was represented by rs13729 from the same ULBP3 gene because of their complete LD in the CHB population (r2 = 1). In total, four SNPs (rs12626864, rs13729, rs1444041, and rs9825563) were genotyped using the Beckman SNPstream genotyping system [63] (link), while one SNP (rs1107877) was genotyped by the PCR-RFLP method (Sau3AI, Takara, China). For 2 UVR-associated SNPs, rs1586360 was not genotyped owing to its low MAF in the CHB populations (MAF < 0.1). Only rs7531583 was genotyped using the same Beckman SNPstream genotyping system.
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5

E. coli Transformation and Plasmid Manipulation

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E. coli competent cells (DH5α and BL21 (DE3)) were bought from Nanjing Vazyme Biotech Co., Ltd. Plasmids, including pET-28a and pUC118 vector, were obtained from Miaoling Biotechnology Co., Ltd. (Wuhan, China). Reaction enzymes (restriction endonucleases including Sau3AI, NcoI, HindIII, and T4 ligase) and DNA/protein markers were procured from Takara Bio (Kusatsu, Japan). All bacterial strains were grown in a commonly used Luria–Bertani (LB) broth at appropriate temperatures.
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6

Cloning the Peace Gene from Genomic DNA

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Cloning of the Peace gene (Peace genomic sequence, GenBank/EMBL/DDBJ accession AB897866) from genomic DNA was carried out following the directions for the Stratagene phage cloning system (Stratagene, USA). Total DNA extracted from V2 petals was partially digested with Sau3AI (TAKARA, Japan). After a partial fill-in reaction, total DNA fragments were ligated with lambda FIX/XhoI partial fill-in treated DNA (Stratagene, USA), and a packaging reaction was carried out using GigaPack Gold (Stratagene, USA). Recombinant phages were selected by plaque hybridization with Peace cDNA (clone cPpP14) as a probe. Plaque hybridization was carried out using an ECL non-radioactive DNA labelling and detection kit (GE Healthcare, UK).
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7

RFLP Analysis for Species Identification

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For RFLP analysis, the COI barcode fragment (Table 1) was digested with HindIII (Takara) to distinguish between D. dentex and other species (Table 3) and with Sau3AI (EnzyQuest) to identify P. pagrus. Additionally, for P. pagrus and related species, the cytb fragment (Table 1) was digested with Sau3AI, and a 1292 bp fragment of the CR (Table 3) amplified with cytbF2 and CRR primers was digested with XbaI (Takara).
The products were run on 1.2% agarose gels stained with Midori Green (Nippon Genetics), and the band sizes were estimated with FastGene® 100 bp DNA Ladder (Nippon Genetics) and 50 bp DNA Ladder (Jena Bioscience GmbH).
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