Genomic DNA of the mutants which showed reduced virulence on cabbage were purified by using a Nucleospin Microbial DNA Kit (TaKaRa, Ohtsu, Japan) and digested with Hind III, Xh I, Sph I, Kpn I, Sal I, Xba I, or Hinc II (TaKaRa). The resultant DNA was ligated with T4 DNA ligase (Ligation-convenience kit, Nippon Gene, Tokyo, Japan), then introduced into E. coli DH5α competent cells. Plasmid DNA was purified from the transformants, and transposon-insertion sites were identified by sequencing with the M13 forward primer. A Pseudomonas Genome DB BLAST search (http://www.pseudomonas.com/blast/setnblast ) was utilized to identify the function of the mutated genes (Fig. 1 ).
Hinc 2
Manufactured by Takara Bio
Sourced in Japan
Hinc II is a type II restriction endonuclease enzyme that recognizes and cleaves the DNA sequence 5'-G^ANTC-3'. It is commonly used in molecular biology applications such as DNA digestion and fragment analysis.
Automatically generated - may contain errors
Lab products found in correlation
Hindiii, by Takara Bio (3 mentions)
Xbai, by Takara Bio (2 mentions)
Nucleospin microbial dna kit, by Takara Bio (2 mentions)
T4 dna ligase ligation convenience kit, by Nippon Gene (1 mentions)
T4 dna ligase, by Nippon Gene (1 mentions)
Xhoi, by Takara Bio (1 mentions)
Dneasy blood and tissue kit, by Qiagen (1 mentions)
Sv total rna isolation system, by Promega (1 mentions)
Ethidium bromide, by Promega (1 mentions)
Ecori, by Takara Bio (1 mentions)
Gel doc system, by Bio-Rad (1 mentions)
Hinfi, by Takara Bio (1 mentions)
Agarose le, by Thermo Fisher Scientific (1 mentions)
Bgiii, by Takara Bio (1 mentions)
Haeiii, by Takara Bio (1 mentions)
Hotstartaq master mix, by Qiagen (1 mentions)
Primer premier version 5, by Premier Biosoft (1 mentions)
6 protocols using hinc 2
1
Identifying Virulence Genes in Pseudomonas Mutants
2
Restriction Enzyme Digestion of PCR Products
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PCR products were ethanol-precipitated and vacuum-dried. Then, products were digested for 1 h with Alu I or Hinc II according to the manufacture's protocol (Takara Bio Inc.), electrophoresed and detected as described above.
3
Mutant Virulence Reduction in Kiwifruit
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Genomic DNA of the mutants that showed reduced virulence on kiwifruit was purified using a Nucleospin Microbial DNA Kit (Takara Bio, Kusatsu, Shiga, Japan) and digested with Hind III, Xho I, Sph I, Kpn I, Sal I, Xba I, or Hinc II (Takara Bio). The resultant DNA was ligated with T4 DNA ligase (Ligation-convenience kit, Nippon Gene, Tokyo, Japan), then introduced into E. coli DH5α competent cells. Plasmid DNA was purified from the transformants, and transposon-insertion sites were identified by sequencing with the M13 forward primer. A Pseudomonas Genome DB BLAST search (http://www.pseudomonas.com/blast/setnblast , accessed on 14 February 2020) was utilized to identify the function of the mutated genes.
4
Prostate Cancer Tissue Analysis Protocol
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Tissue samples were collected with approval of the Ethical Committee of the Piedmont Region, Italy, and written informed consent from patients undergone radical prostatectomy for localized prostate cancer62 (link). Total RNA was extracted from snap frozen tumour samples (n=24) using SV Total RNA Isolation System (Promega) and analysed by RT–qPCR or strand-specific RT–qPCR. Primers (paRNA, CDH1 −282F and −171R; CDH1 mRNA, CDH1 +2497F and +2619R) are shown in Supplementary Table 2 . For genotyping by restriction fragment length polymorphism (RFLP) analysis or sequencing, genomic DNA was extracted from two 10-μm sections of formalin-fixed paraffin embedded (FFPE) prostate tumour specimens (n=47) using DNeasy Tissue and Blood kit (Qiagen). Genomic DNA was amplified by PCR along with positive (Male DNA Universal Reference) and negative control (No template) reactions using primers (CDH1 −272F and −82R). PCR products (5 μl for cell lines and 15 μl for FFPE samples) were digested with HincII (TaKaRa) and analysed by agarose gel electrophoresis. SNP analysis in cell lines was performed by RFLP and sequencing. The relationship between SNP rs16260 and recurrence-free survival was examined in a patient cohort with available pathological and clinical follow up data24 (link).
5
Bacterial 16S rRNA RFLP Analysis
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Based on the clustering patterns, RFLP was performed using one sample from each cluster. Before RLFP, the 16S rRNA region was amplified using the universal bacterial primers (27F: 5′-AGAGTTTGATYMTGGCTCAG-3′ and 1492R: 5′-GGTTACCTTGTTACGACTT-3′, both to a final concentration of 0.5 mM [37 (link)], 25 µL of Hot Star Taq master mix (QIAGEN, Hilden, Germany), ≤20 ng of DNA and RNase free water up to 50 µL. The reaction was amplified in Biometra TAdvanced Thermal Cycler (Analytik, Jena, Germany) as previously described [38 ]. Restriction enzymes used were ECORI, BGII, BGIII, HinfI, AluI, HaeIII, HincII, HindIII, SacI, SmaI (Takara, Japan) according to instructions of the manufacturer. The RLFP reactions were carried out in 10 µL volume containing 0.5 µL enzyme, 1 µL buffer, 2.5 µL, an additional 1 µL Bovine Serum Albumin (BSA) for SmaI and nuclease-free water up to 10 µL. RLFP samples were then incubated at 37 °C except for SmaI samples, which were incubated at 30 °C. After incubation, 1 µL of stop solution was added to the samples, which were then subjected to electrophoresis in 1.2% agarose (Agarose-LE, Ambion®, Pittsburgh, PA, USA), stained with ethidium bromide (Promega, Madison, WI, USA), and visualized using Bio-Rad gel doc system (Bio-rad, Gel Doc tm X. R. System 170–8170, Montréal, QC, Canada).
6
Genetic Polymorphism Analysis of Surfactant Proteins
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Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was used to analyze SP-A, SP-B, and SP-D genetic polymorphisms. PCR primers were designed using Primer Premier version 5.0 software (Premier Biosoft, Palo Alto, CA) and synthesized by Shanghai Sangon Biological Engineering Technology Co., Ltd. (Shanghai, China). The primer sequences and their lengths are shown in Table 1 . PCR reaction was carried out in 20-μl volume, containing 10 μl×PCR PLUS MIX (DBI), 1.2 μl DNA templates, 0.5 μl upstream primer, and 0.5 μl downstream primer. PCR conditions were: 95°C initial denaturation for 5 min, 95°C denaturation for 30 s, 60°C annealing for 45 s, and 72°C extension for 50 s. After 35 cycles, the final extension was at 72°C for 10 min. PCR amplification products (6 ml) were digested with 4 U Fsp I (Toyobo) in a 15-l volume and incubated overnight at 37°C. A volume of 5 μl of the digested product was mixed with 3 μl 6×buffer solution and analyzed on 3% agarose gel, followed by UV photography to record the results. Locus-specific restriction enzymes HincII, XspI, and Ase I (TaKaRa) were also used to validate PCR products from different loci, and agarose gel electrophoresis was employed to analyze and record the results. All genotypes were finally confirmed by sequencing using a DNA sequencer (ABI370).
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