Dna marker

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DNA marker is a laboratory tool used to determine the size of DNA fragments during gel electrophoresis. It contains a mixture of DNA fragments of known sizes, which serve as a reference for comparing the size of unknown DNA samples.

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100 bp DNA ladder marker (5 citations) λ DNA/HindIII marker (4 citations) DNA molecular weight marker (100 bp, (4 citations) DNA and protein markers (3 citations) DNA molecular weight marker (3 citations) DNA marker and protein marker (3 citations) Molecular markers for DNA (2 citations) Ladder 100 bp Plus Opti-DNA Marker (2 citations) GeneRuler 1 Kb DNA Ladder molecular weight marker (2 citations) Lambda DNA/HindIII molecular weight marker (2 citations)

12 protocols using dna marker

Serotyping Streptococcus suis using Multiplex PCR and Agglutination Tests

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The amplified products of S. suis serotypes 1 versus 14 and 2 versus 1/2 showed similar sizes by multiplex PCR (Fig. 2). Therefore, they were subsequently differentiated by slide agglutination combined with precipitation test. The antisera for testing serotype 1 and 2 were prepared in NIAH, Thailand [25 ], while the antiserum for serotype 14 was obtained from the Staten Serum Institute, Copenhagen, Denmark. Slide agglutination test was performed by mixing 1–2 colonies of S. suis with a drop of specific antiserum on a slide. Agglutination between the homologous antigen and antiserum should be observed. The precipitation test was performed in capillary tubes as described elsewhere [28 ]. Briefly, the microhematocrit tube (Vitrex Medical, Denmark) was dipped into the tube containing specific antiserum until the antisera filled up to 1 cm. Subsequently, the same volume of extracted antigen of the suspected S. suis serotype was also filled and the precipitation line was observed within 15 min.Fig. 2

Amplification of multiplex PCR of reference S. suis. Lane 1 and 11, DNA marker
(Fermentas, USA); Lane 2, Negative control (DW); Lane 3, serotype 8; Lane 4, serotype 1; Lane 5,
serotype 14; Lane 6, serotype1/2; Lane 7, serotype 2; Lane 8, serotype 7; Lane 9, serotype 9; Lane
10, mixture of serotypes 1, 2, 7, 9, and 14

Thongkamkoon P., Kiatyingangsulee T, & Gottschalk M. (2017). Serotypes of Streptococcus suis isolated from healthy pigs in Phayao Province, Thailand. BMC Research Notes, 10, 53.

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Ovarian Cancer SKOV3 Cells Analysis

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TRIzol reagent was purchased from Gibco (Grand Island, NY, USA), RT-PCR kit was purchased from Promega Corp. (Madison, WI, USA), heat-resistant DNA polymerase and ELF5 gene primer was compounded by Sangon Biotech Co., Ltd. (Shanghai, China). DNA endonuclease, DNA ligase and DNA marker were purchased from Fermentas (Glen Burnie, MD, USA). DNA gel extraction kit and agarose were purchased from Tiangen Biotech (Beijing) Co., Ltd. (Beijing, China). Medium extraction kit was purchased from Axygen Biosciences Co., Ltd. (Hangzhou, China). Absolute ethyl alcohol, isopropanol, glycerol and anhydrous calcium chloride were purchased from Sinopharm Chemical Reagent Co., Ltd. (Beijing, China). Human ovarian carcinoma SKOV3 cells were purchased and stored by the Laboratory of Obstetrics and Gynecology Department of the Affiliated Hospital of Xuzhou Medical University. Methyl thiazolyl tetrazolium (MTT) was the product of Sigma-Aldrich (St. Louis, MO, USA). Liptapfector lipofectin transfection reagent was provided by Beyotime Biotech (Jiangsu, China). Transwell chamber and ELF5 primary antibody (dilution, 1:1,000; cat. no. AM254515) was provided by Chemicon (Temecula, CA, USA). Escherichia coli DH5α was purchased and stored by the Laboratory of Obstetrics and Gynecology Department of the Affiliated Hospital of Xuzhou Medical University.

Yan H., Qiu L., Xie X., Yang H., Liu Y., Lin X, & Huang H. (2017). ELF5 in epithelial ovarian carcinoma tissues and biological behavior in ovarian carcinoma cells. Oncology Reports, 37(3), 1412-1418.

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Reagents and Suppliers for Molecular Experiments

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All reagents used in the present study were purchased from Sigma (Missouri, USA) unless otherwise indicated. The oligonucleotides were synthesized by Biomatik Company (Wilmington, USA). Restriction enzymes were purchased from Takara (Kyoto, Japan); plasmid DNA extraction and gel purification kits were obtained from iNtRON (Daejoen, Korea); T4 ligase, DNA marker, and IPTG were purchased from Fermentas (Opelstr, Germany); protein marker was purchased from Cinaclone (Tehran, Iran); and Ni-NTA agarose resin was obtained from Qiagen (Valencia, USA).

Faramarzi R, & Dolatabadi S. (2017). Expression and purification of recombinant HTLV-I/-II linear epitopes antigen and its application for screening of suspected patients. Iranian Journal of Microbiology, 9(1), 43-49.

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Recombinant Protein Expression in E. coli

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Host strain E. coli BL 21 Star^TM (DE3) and vector pET-22b(+) were from Invitrogen (Carlsbad, CA, USA). DNA polymerase, DNA marker, T4 DNA ligase, and restriction endonucleases Nde I and Hind III were from Fermentas (Waltham, MA, USA). A polymerase chain reaction (PCR) amplification kit (including PCR buffer and dNTP mix) was obtained from Takara (Shiga, Japan). The plasmid mini kit I and PCR product recovery kit were purchased from Omega Bio-Tek (Norcross, GA, USA). Uric acid standard was from Sigma-Aldrich (St. Louis, MO, USA). All other reagents were of analytical grade.

Xie G., Yang W., Chen J., Li M., Jiang N., Zhao B., Chen S., Wang M, & Chen J. (2016). Development of Therapeutic Chimeric Uricase by Exon Replacement/Restoration and Site-Directed Mutagenesis. International Journal of Molecular Sciences, 17(5), 764.

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Plasmid DNA Purification and PCR Amplification

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HiPure Gel Pure DNA Mini Kit (Magen) was used to purify PCR fragments and Plasmid Miniprep Kit (Zoman) was used to isolate plasmid DNA. All the restriction enzymes, alkaline phosphatase, T4 DNA ligase and DNA marker were purchased from Thermo. High-fidelity PCRs and general PCRs were performed with KOD-Plus-Neo (TOYOBO) and 2 × Hieff™ PCR Master Mix (YEASEN), respectively.

Bu Q.T., Yu P., Wang J., Li Z.Y., Chen X.A., Mao X.M, & Li Y.Q. (2019). Rational construction of genome-reduced and high-efficient industrial Streptomyces chassis based on multiple comparative genomic approaches. Microbial Cell Factories, 18, 16.

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Evaluating the Anticancer Potential of SI-4650 in A549 Cells

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SI-4650 (N-(3-{[3-(dimethylamino)propyl]amino}propyl)-8-quinolinecarboxamide) was purchased from SPECS (Zoetermeer, The Netherlands), DMSO, 3-(4,5-dimethylthiazol)-2,5-diphenyltetrazolium bromide (MTT), Spermine, horseradish peroxidase, luminol, 1,7-Diaminoheptane were purchased from Sigma (St. Louis, MO), foetal calf serum was purchased from Gibco (Grand Island, NY), Turbofect transfection reagent, DNA marker and protein marker were purchased from Thermo Fisher Scientific (Waltham, MA), Transwell chamber was purchased from Corning (Corning, NY), FITC Annexin V Apoptosis Detection Kit was purchased from BD Biosciences Inc. (San Jose, CA). The primary antibodies were all obtained from Santa Cruz Biotechnologies: p62(catalog no. 18420-1-AP), p21(catalog no. 103551-AP), CyclinB1(catalog no. 55004-1-AP), LC3(catalog no. 12741 s), Bax(catalog no. 50599-2-Ig), Bcl-2(catalog no. 12789-1-AP). All other chemicals used in the experiments were commercial products of reagent grade.
The A549 cell line was obtained from the Cell Bank of China (Wuhan, China). Cells were cultured in RPMI-1640 supplemented with 10% foetal calf serum, 5% CO₂ at 37 °C.

Sun L., Yang J., Qin Y., Wang Y., Wu H., Zhou Y, & Cao C. (2019). Discovery and antitumor evaluation of novel inhibitors of spermine oxidase. Journal of Enzyme Inhibition and Medicinal Chemistry, 34(1), 1140-1151.

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Recombinant Protein Expression Workflow

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The chemicals were generally purchased from Sigma-Aldrich (St. Louis, USA). Tryptone and yeast extract were from Merck (Darmstadt, Germany). Restriction enzymes, DNA marker, T4 DNA ligase, and DNA polymerase were purchased from Thermo Fisher Scientific (Waltham, USA). Protein marker was purchased from SinaClon (Tehran-Iran). The pET26b vector and E. coli strains including DH5α and BL21(DE3) were from Novagen (Madison, USA). The kits for PCR product purification and plasmid extraction were from GeneAll (Seoul, Korea). The Ni–NTA protein purification resin was purchased from Qiagen (Hilden, Germany).

Ghadikolaei K.K., Sangachini E.D., Vahdatirad V., Noghabi K.A, & Zahiri H.S. (2019). An extreme halophilic xylanase from camel rumen metagenome with elevated catalytic activity in high salt concentrations. AMB Express, 9, 86.

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In Vitro RNA Synthesis and Purification

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Oligonucleotides were synthesized by Genecreate Company, DNA purification kits were from Axygen, and Ni-NTA resin was from Qiagen. Preparative Superdex S200 for gel filtration was from GE Healthcare. The Gibson Assembly kit, T4 RNA ligase I, recombinant inorganic pyrophosphatase, rNTPs, DNase I, apyrase, T7 RNA polymerase (50 U/μL), low-range ssRNA ladder, and Monarch RNA Cleanup kit were from New England Biolabs. 2’-Fluoro-dNTPs, N6-methyl adenosine-5′-triphosphate (m6ATP), and 5-methylcytosine-5’-triphosphate (5mCTP) were from TriLink BioTechnologies. A reverse transcriptase kit and PrimeSTAR Max DNA polymerase were from TaKaRa. RiboLock RNase inhibitor and RNase A (10 mg/mL) were from Thermo Scientific. J2 monoclonal antibody (mAb) was from English & Scientific Consulting. An Immobilon-Ny+ membrane was from Millipore. X-ray film was from Kodak. DNA marker and an electrochemiluminescence (ECL) reagent were from Thermo Scientific. A high-performance liquid chromatography (HPLC) polymeric reversed-phase media (PLRP-S) column was from Agilent.

Xia H., Yu B., Jiang Y., Cheng R., Lu X., Wu H, & Zhu B. (2022). Psychrophilic phage VSW-3 RNA polymerase reduces both terminal and full-length dsRNA byproducts in in vitro transcription. RNA Biology, 19(1), 1130-1142.

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Cefiderocol Antimicrobial Susceptibility Testing

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Cefiderocol (CEF) was purchased from ChemScene LLC (USA). Mueller–Hinton Broth (MHB), Luria-Bertani (LB) Broth Powder, CA-MHB Broth Power, M9 Broth Powder and Agar powder were purchased from Affymetrix. Tryptone and Yeast extract were purchased from Oxoid Ltd. (UK). dNTPs, pfu DNA polymerase, Taq DNA polymerase, and DNA marker were purchased from Thermo Fisher Scientific Ltd. Restriction endonucleases (BamH I, Hind III, Sac I) were purchased from TAKARA. Plasmid-Mini-prep Kit and Genomic DNA Purification Kit were purchased from Thermo Fisher Scientific Ltd. All other chemicals were purchased from Sigma-Aldrich unless specified.

Wang C., Xia Y., Wang R., Li J., Chan C.L., Kao R.Y., Toy P.H., Ho P.L., Li H, & Sun H. (2023). Metallo-sideromycin as a dual functional complex for combating antimicrobial resistance. Nature Communications, 14, 5311.

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Generation and Characterization of CAR T Cells

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Chemical and biological products include the following:

The plasmids: pcDNA 3.1+CAR gene (genscript), Vector pFUGW (from addgen, code 14883), pCMV-dR8.2dvpr (from addgen, code 8455), and pCMV-VSV-G (from addgen, code 8454)

Cell culture medium DMEM, FBS, Opti-MEM (Gibco); penicillin/streptomycin, Trypsin, Trypan blue (Pan Biotech), Ficoll-PaqueTM (GE Healthcare)

T-CD3 cell separator kit; TexMAXS Medium, IL-2; T Cell TransAct were obtained from Miltenyi Biotec

Human IFN gamma ELISA Kit; Human IL-2 ELISA Kit (Abcam)

The enzyme T4 DNA ligase; EcoRI and BamHI (BioLabs); DNA marker (Thermo); Plasmid DNA MIDI KIT (QIAGEN); Gel Extraction Kit (QIAGEN); Lipofectamine™ 3000 Transfection Reagent, P3000™ Reagent (Invitrogen).

Easy-BLUE™ Total RNA Extraction Kit (iNtRON), real-time Luna universal one-step RT-qPCR kit (Biolabs); Total DNA extraction kit (QIAgen); PCR master mix (Promega)

Gene test primer sequences are presented in Table 1

La H.T., Tran D.B., Tran H.M, & Nguyen L.T. (2021). Third-Generation Anti-CD47-Specific CAR-T Cells Effectively Kill Cancer Cells and Reduce the Genes Expression in Lung Cancer Cell Metastasis. Journal of Immunology Research, 2021, 5575260.

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