Tianamp bacteria dna kit

We purchased TIANamp Bacteria DNA Kits from Tiangen Biotech Co., Ltd.(Beijing, China). Lysozyme and agarose were purchased from Beyotime Biotechnology Co., Ltd.(Shanghai, China). The isothermal amplification kits and Malachite Green were purchased from Huidexin Bio-technology Co., Ltd.(Tianjin, China). qPCR Mix, pMD19-T Vector and DH5α were purchased from Takara Biomedical Technology Co., Ltd. (Beijing, China). The The swabs were purchased from Copan Diagnostics, Inc. (Lombardy, Italy). Todd Hewitt medium, gentamicin and nalidixic were obtained from Qingdao Hi-Tech Industrial Park Haibo Biotechnology Co., Ltd(Qingdao, China).

Firstly sterilised normal saline was used to adjust the suspension to 1 McFarland turbidity. The bacterial suspension was subsequently centrifuged at 10 000 rpm for one minute at 4 °C, and the supernatant was discarded. Ultimately DNA was extracted from S. pneumoniae strains using TIANamp Bacteria DNA Kits (TIANGEN, China) according to the manufacturer's instructions.

Total DNA from the Salmonella isolates was extracted using a TIANamp Bacteria DNA kits (Tiangen, Beijing, China) and then subjected to whole genome sequencing. The library was constructed using a Next^® Ultra™ DNA Library Prep kit (New England Biolabs, Ipswich, UK) according to the manufacturer’s protocol, and 250-bp paired-end reads were obtained from an Illumina Hiseq2500 platform (Bionova Biotech Co., San Diego, CA, USA). For each isolate analyzed by Whole-Genome Sequencing, at least 100-fold coverage of raw reads were collected. A draft assembly of the sequences was generated using SOAPdenovo version 2.04 (http://soap.genomics.org.cn/soapdenovo.html).
Assembled data, S. 4,[5],12:i:- strain SO4698-09 (RefSeq NZ_LN999997) were subjected to SNP calling and reference-based phylogeny tree building using CSI Phylogeny 1.4 [22 (link)] with SO4698-09 as the reference genome and default parameters for SNP filtering and pruning.

LF-HY01 was isolated from traditional fermented yak yoghurt using the serial dilution and spread plate method and cultured at 37 °C for 48 h in MRS both (BD, Franklin Lakes, NJ, USA); the MRS solid medium contained 1.5% agar. The cell morphology was observed after gram staining, and the genus was analyzed using 16S rDNA sequence and phylogenetic tree. Firstly, the genome DNA from LF-HY01 was extracted by TIANamp bacteria DNA kit (Tiangen, Beijing, China), its 16S rDNA was amplified with S1000 Thermal Cycler (Bio-Rad, Hercules, CA, USA), and the amplification product was tested by using 1.5% agarose gel electrophoresis. Finally, the remaining amplification product was sent to sequence (BGI, Shenzhen, Guangdong, China). Finally, based on 16S rDNA sequence of LF-HY01 and reference strains, a phylogenetic tree was built by using MEGA 5.05. During the experiment, LF-HY01 was activated and cultured at 37 °C for 18 h in MRS both.

H. pylori colonization and immune responses in the stomachs were detected by quantitative real-time PCR. The bacterial genome was extracted from the mouse stomachs with a TIANamp Bacteria DNA Kit (TIANGEN, DP302) and dissolved in 200 μl deionized water. Then, 2 μl of H. pylori 16S rDNA was analyzed. For the quantitative real-time PCR, the target gene with a concentration gradient was set as standards, together with positive and negative controls. Two replicates were used for each sample. The detection result multiplied by 100 was regarded as the H. pylori colonization level in a mouse stomach, and the data are presented as log10 values (sense primer, 5′-TTTGTTAGAGAAGATAATGACGGTATCTAAC-3′, anti-sense primer, 5′-CATAGGATTTCACACCTGACTGACTATC-3′, H. pylori 16s probe, FAM-CGTGCCAGCAGCCGCGGT-TAMRA). Total RNA from the gastric single cells harvested above was extracted by TRIzol and reverse transcribed into cDNA with a PrimeScript TM 1st Strand cDNA Synthesis Kit (Takara). Then, the expression levels of interferon γ (IFN-γ) and interleukin 17A (IL-17A) were determined by quantitative real-time PCR with SYBR green staining (IFN-γsense primer, 5′-GATCCTTTGGACCCTCTGACTT-3′, IFN-γanti-sense primer, 5′- TGACTGTGCCGTGGCAGTAA-3′, IL-17A sense 5′-CTCCAGAAGGCCCTCAGACTAC-3′, IL-17A anti-sense 5′-GGGTCTTCATTGCGGTGG -3′).

The B. coagulans L01 strain was cultured in LB medium for 12 h. Its genomic DNA was extracted by using TIANamp Bacteria DNA Kit (TIANGEN, Beijing) and then was used as the template DNA of PCR amplication. The primers used for cloning the araA gene were 5′-CGCGGATCCGATGTTGAAAATAAAAGA-3′ (forward primer) and 5′-CCGGAATTCTGTTAAAGAAGTGCATC-3′ (reverse primer). The underlined sequences represent restriction sites BamH I and EcoR I respectively. The PCR product was ligated with pEASY-Blunt cloning vector. The resulting recombinant plasmid was sequenced by BGI Tech. (Shanghai, China). Then, both the recombinant cloning plasmid and the expression vector pETDuet-1 were digested with BamH I and EcoR I, and the araA gene was cloned into the multiple cloning sites of pETDuet-1 to generate the recombinant expression plasmid, pETDuet-araA. Finally, the plasmid was transformed into E. coli BL21 (DE3) for expression.

Restriction enzyme digestion, ligation, gel electrophoresis and other general molecular techniques were performed as previously described [19 ]. A. caldus genomic DNA was isolated using the TIANamp Bacteria DNA Kit (TIANGEN). Plasmids were isolated using plasmid mini kit I (Omega Bio-Tek). DNA fragments were extracted from agarose gels using a gel extraction kit (Omega Bio-Tek). DNA polymerase, restriction enzymes and T4 DNA ligase were purchased from TaKaRa, and primers were generated by Invitrogen.