The NSC34 cells were purchased by Tebu-BIO [Magenta, (MI) Italy]. Cells were used between 5–20 passages. Cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) in the presence of 10% inactivated fetal bovine serum and penicillin/streptomycin at 37°C, 5% CO2 and 95% air, in a cell culture incubator. SH-SY5Y cells were purchased and grown as previously published (Guida et al., 2015a (link)). Methylmercury (II) chloride (MeHg) (cod: 442534 stock solution 100 mM) and Necrostatin-1 (Nec) (cod. N9037; stock solution 20 mM) were purchased from Sigma both obtained from Sigma–Aldrich (St. Louis, MO, United States) and were dissolved as previously reported (Guida et al., 2018 (link)). Culture media and sera were purchased from Invitrogen (Milan, Italy). All chemicals were diluted in cell culture medium. Synthetic oligonucleotides were from Eurofins Genomics. For Nec experiments NSC34 cells were pre-incubated for 2 h in full medium with the drug at 1, 5, 10, and 20 μM, followed with MeHg 100 nM for 24 h. For MTT and LDH assays, cells were plated in 24 well plates at a density of 1 × 104 cells/well; for qRT-PCR, they were plated in 60 mm plates at a density of 5 × 104 cells/plate; for Western blot, Immunoprecipitation and ChIP, they were plated in 100 mm plates at 6 × 106 cells/plate.
Synthetic oligonucleotides
Manufactured by Eurofins
Sourced in Germany, United States
Synthetic oligonucleotides are short, single-stranded DNA or RNA molecules that are artificially synthesized in a laboratory. They serve as essential tools for various genetic and molecular biology applications, including DNA sequencing, gene expression analysis, and molecular diagnostics.
Automatically generated - may contain errors
Lab products found in correlation
Anti β actin, by Merck Group (2 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (2 mentions)
Megascript t7 transcription kit, by Thermo Fisher Scientific (2 mentions)
Nucleospin mirna kit, by Macherey-Nagel (2 mentions)
Restriction enzyme, by New England Biolabs (2 mentions)
Dna modifying enzymes, by New England Biolabs (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
T4 dna ligase, by New England Biolabs (1 mentions)
Lysozyme, by Merck Group (1 mentions)
Qiaquick gel extraction kit, by Qiagen (1 mentions)
Qiaprep spin miniprep kit, by Qiagen (1 mentions)
Luciferase assay system, by Promega (1 mentions)
Bovine serum albumin (bsa), by Thermo Fisher Scientific (1 mentions)
Ampicillin, by Thermo Fisher Scientific (1 mentions)
Kanamycin, by Merck Group (1 mentions)
Ortho nitrophenyl β galactoside, by Merck Group (1 mentions)
Qiaquick nucleotide removal kit, by Qiagen (1 mentions)
T4 polynucleotide kinase, by New England Biolabs (1 mentions)
Penicillin streptomycin, by Merck Group (1 mentions)
Trypsin, by Merck Group (1 mentions)
29 protocols using synthetic oligonucleotides
1
Neuroprotective Effects of Necrostatin-1 against Methylmercury Toxicity
2
Biomass Pretreatment for Bioenergy
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All commercially available substrates were purchased from Merck, Carbosynth and Megazyme. The synthetic oligonucleotides were from Eurofins (Italy). The pretreated biomass of A. donax used in this study was derived from a pretreatment step by PROESA® technology of the Chemtex Group. C. cardunculus used in this study was provided by Novamont. Both biomasses were provided in the frame of the project “PON 01_0966 ENERBIOCHEM”.
3
Purification and Detection of Etx Pore
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Chemicals were purchased from Sigma, UK, unless otherwise stated. The polyclonal antibody against Etx mutant Y30A-Y196A was raised in rabbits as described in ref. 51 (link). After electrophoretic separation purified Etx pore was detected by SimplyBlue staining (Invitrogen Ltd, Paisley, UK). Protein concentrations were determined using the Pierce™ BCA Protein Assay Kit (Fisher Scientific Ltd, Loughborough, UK). Synthetic oligonucleotides were sourced from Eurofins Genomics, Germany.
4
Synthetic Oligonucleotide Sourcing Protocol
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Synthetic oligonucleotides used in this study were purchased from Eurofins Genomics (Ebersberg, Germany) and are shown in Table S1 .
5
Molecular Cloning Reagents and Protocols
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Kanamycin, lysozyme, and ortho-Nitrophenyl-β-galactoside (ONPG) were purchased from Sigma-Aldrich Corporation (St. Louis, MO). Ampicillin and bovine serum albumin (BSA) were bought from Fisher Scientific (Fairlawn, NJ). Isopropyl-β-D-thiogalactopyranoside (IPTG) was obtained from Anatrace, Inc (Maumee, Ohio). All restriction enzymes, T4 DNA ligase, and T4 polynucleotide kinase were purchased from New England Biolabs (Beverly, MA). Pfu DNA polymerase was obtained from Stratagene, Inc. (La Jolla, CA). All synthetic oligonucleotides were purchased from Eurofins Genomics (Huntsville, AL). Plasmid and DNA fragment cleaning kits including QIAprep Spin Miniprep Kit, QIAquick Gel Extraction Kit, and QIAquick Nucleotide Removal Kit were obtained from QIAGEN, Inc. (Valencia, CA). Luciferase Assay System was bought from Promega Corporation (Madison, WI).
6
Sphingosine-1-Phosphate Signaling Pathway
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The following materials were purchased: LHC Basal, LHC-8 w/o gentamicin culture media from Gibco (CA, USA; cat n°12679-015); FBS (cat n°ECS0180L), MEM (cat n°ECB2071L) and DMEM (cat n°ECM0728L) from Euro Clone Life Science Division (Milan, Italy); protease inhibitors from Roche Italia (Monza, MB, Italy); penicillin/streptomycin (cat n°ECB3001D) and Trypsin (cat n°ECB3051D) from Sigma-Aldrich (Burlington, MA, USA; Quick Start™ Bradford Dye Reagent (cat n°ECL170-S061) and Clarity™ Western ECL Blotting Substrates from BioRad (Milan, Italy); SYBR Green system from Takara (Kusatsu, Japan; cat n°RR420L); ReliaPrep™ Miniprep RNA extraction System (cat n°Z6011) and GoScript Reverse Transcription Mix (cat n°A2791) from Promega (Madison, WI, USA); synthetic oligonucleotides from Eurofins Genomics (Ebersberg, Germany). Methanol, acetonitrile, ammonium formate, acetic acid, potassium hydroxide and formic acid (all analytical grade) were supplied from Merck (Darmstadt, Germany). Water was MilliQ grade. Sphingosine 1-phosphate standard (cat n°8604928) was purchased by Avanti Polar Lipids (Alabaster, AL, USA). Primary antibodies: anti-β-actin (Sigma, Burlington, MA, USA; cat n°A5316, clone AC-74), anti-Spns2 (Gene Tex, Irvine, CA, USA; cat n°GTX45115). The secondary antibodies were from Jackson Laboratories (Bar Harbor, ME, USA). All reagents were of the maximal available purity degree.
7
Multiplex MLPA Analysis of C19orf12 Gene
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A total of five C19orf12‐specific probes targeting the promoter (n = 1), the coding exons (n = 2), the 3'UTR (n = 1), and a region approximately 1 Kb downstream of the gene (n = 1) were utilized for multiplex ligation‐dependent probe amplification (MLPA) analysis by the Beetz Lab (Dept of Clinical Chemistry, Jena University Hospital, Germany). Five reference probes were directed against regions on distinct other chromosomes. MLPA probe design followed the guidelines provided by MRC‐Holland (The Netherlands) at www.mlpa.com . The corresponding synthetic oligonucleotides were purchased from MWG‐Eurofins (Ebersberg, Germany). MLPA reactions utilized reagents from MRC‐Holland. Analysis of data was done as described previously (Beetz et al., 2006 ).
8
Reagents and Antibodies Procurement
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All reagents were obtained from Sigma-Aldrich if not otherwise specified. Synthetic oligonucleotides were obtained from Eurofins (Eurofins MWG Operon, Huntsville, AL, USA), purified by HPLC, as listed in Supplementary Table 5 . Antibodies are listed in Supplementary Table 6 .
9
Recombinant Protein Expression in E. coli
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Escherichia coli strain DH5α and BL21 (DE3) were used as a host for cloning and expression of recombinant proteins, respectively. Escherichia coli cells were grown in lysogeny broth (LB) medium at 37°C. Synthetic oligonucleotides, obtained from Eurofins Genomics, are listed in Supplementary Table 1 .
10
Oligonucleotide Synthesis and Purification
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All reagents were obtained from Sigma–Aldrich (St. Louis, MO, United States) if not otherwise specified. Synthetic oligonucleotides were obtained from Eurofins MWG Operon (Huntsville, AL, United States) and purified by HPLC.
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