Eco32i

Manufactured by Thermo Fisher Scientific

Sourced in Germany

The Eco32I is a compact and durable refrigerated incubator shaker from Thermo Fisher Scientific. It provides a controlled environment for cell culture, microbiology, and other applications requiring temperature and agitation. The Eco32I offers a temperature range of 5°C above ambient to 65°C, with digital PID temperature control and a user-friendly display.

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Lab products found in correlation

8 protocols using eco32i

Molecular Cloning and Protein Expression Techniques

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AccuTaq LA DNA Polymerase was from Sigma (Sigma-Aldrich, St. Louis, MO, USA), the T4 DNA Ligase from Promega (Promega, Switzerland), T4 Polynucleotide Kinase, Shrimp Alkaline Phosphatese, restriction enzymes Acc65I, AccI, SmaI, Hind II, XhoI, NheI, Cfr9I, XbaI, XmaJi, Cfr10I, Eco32I from Fermentas (Fermentas, Payerne, Switzerland), Taq-Polymerase and LipofectaminTM 2000 from Invitrogen (Invitrogen, Switzerland), PfuUltra Hotstar DNA Polymerase from Agilent (Agilent, Basel, Switzerland), and the Big dye Terminator from Applied Biosystems (Applied Biosystems, Muttenz, Switzerland). The p-38 MAPK inhibitor (SB203580) was obtained from Jena Bioscience (Jena Bioscience GmbH, Jena, Germany). Antibodies against β-actin (sc-47778, 1:1000), PGC-1α (sc-518025, 1:1000), and MEF2 (1:1000) were from Santa Cruz Biotechnology (Dallas, TX, USA), against OCTN2 (ab180757, 1:1000) from Abcam (Cambridge, UK) and the control IgG from Santa Cruz Biotechnology (Dallas, TX, USA).
The DNA purification kit NucleoSpin^®Tissue and the NucleoBond^®AX Plasmid Purification kit were from Macherey-Nagel (Düren, Germany), the QIAquick Gel Extraction Kit from Qiagen (Hilden, Germany), the Dual-Luciferase^® Reporter 1000 Assay System from Promega (Promega, Switzerland), and Micro Bio-spin chromatography columns and the Bio-Rad protein assay from Bio-Rad (Bio-Rad Laboratories AG, Cressier, Switzerland).

Novakova K., Török M., Panajatovic M., Bouitbir J., Duong F.H., Handschin C, & Krähenbühl S. (2022). PGC-1α and MEF2 Regulate the Transcription of the Carnitine Transporter OCTN2 Gene in C2C12 Cells and in Mouse Skeletal Muscle. International Journal of Molecular Sciences, 23(20), 12304.

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Chromosomal Integration Site of Brucella Phage

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To determine the chromosomal integration site of the phage, gDNA of B. inopinata BO1 and B. abortus S19lysBiPBO1 was isolated using the DNA mini preparation kit (Qiagen, Hilden, Germany). The DNAs were digested with several restriction endonucleases (Bsp143I, DpnI, DraI, Eco32I, HindIII) according to the manufacturer's recommendations (Fermentas, St. Leon Roth, Germany). Restriction fragments of each digest were treated with T4 ligase (Fermentas) and used as template in a PCR reaction applying outward facing primers deduced from the coding region of the BiPBO1 integrase gene (Table S5). PCR products were purified (QIAquick PCR purification kit, Qiagen) and sequenced. The obtained nucleotide sequences of the amplicons were compared with whole genome sequences of the respective strains. To study the BiPB01 integration site in other Brucella strains, a Multiplex-PCR was developed. Primers deduced from the Brucella chromosome left and right of the BiPB01 integration site provided information on the presence of foreign DNA at this position. Combinations of these primers with primers deduced from the BiPB01 genome were used to detect BiPB01-related prophage DNA. All primers are listed in Supplementary Material Table S5.

Hammerl J.A., Göllner C., Al Dahouk S., Nöckler K., Reetz J, & Hertwig S. (2016). Analysis of the First Temperate Broad Host Range Brucellaphage (BiPBO1) Isolated from B. inopinata. Frontiers in Microbiology, 7, 24.

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Genomic DNA Extraction and Southern Blot

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Genomic DNA was extracted using the gDNA Midi Kit (Zymo Corp, Irvine, CA). 7–15 µg genomic DNA from each iPSC line were digested overnight with Eco32I or EcoO109I (Fermentas) and resolved by electrophoresis on 0.8–1.0% agarose gels at 16–22 V for 6–10 h. DNA was transferred using the Whatman kit (Schleicher and Schuell, Keene, NH) and nitrocellulose membrane (Hybond-N; GE Healthcare, Piscataway, NJ). The Turboblotter transfer apparatus (Schleicher and Schuell) was used following the manufacturer's instructions. A DIG-labeled GFP probe generated by the DIG High Prime Labeling and Detection Starter Kit II (Roche, Indianapolis, IN) was then hybridized to the membrane following the manufacturer's instructions.

Zhao C., Farruggio A.P., Bjornson C.R., Chavez C.L., Geisinger J.M., Neal T.L., Karow M, & Calos M.P. (2014). Recombinase-Mediated Reprogramming and Dystrophin Gene Addition in mdx Mouse Induced Pluripotent Stem Cells. PLoS ONE, 9(4), e96279.

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Phage DNA Extraction and Restriction Analysis

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The aliquots of phage suspension (10¹¹–10¹² PFU/mL) were subjected to phenol/chloroform extraction and ethanol precipitation, as described by Carlson and Miller [22 ]. The isolated phage DNA was subsequently used in restriction analysis, for PCR, or it was subjected to genome sequencing. The restriction digestion was performed with BamHI, Bsu15I, Csp6I, DraI, EcoRII, Eco32I, HhaI, MboI, MfeI, NdeI, and RsaI restriction endonucleases (Thermo Fisher Scientific, Vilnius, Lithuania), according to the supplier’s recommendations. The DNA fragments were separated by electrophoresis in a 0.8% agarose gel containing ethidium bromide. A restriction analysis was performed in triplicate to confirm the results.

Šimoliūnas E., Šimoliūnienė M., Kaliniene L., Zajančkauskaitė A., Skapas M., Meškys R., Kaupinis A., Valius M, & Truncaitė L. (2018). Pantoea Bacteriophage vB_PagS_Vid5: A Low-Temperature Siphovirus That Harbors a Cluster of Genes Involved in the Biosynthesis of Archaeosine. Viruses, 10(11), 583.

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Phage DNA Characterization by Enzymatic Assays

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Aliquots (100–150 μl) of phage suspensions (10¹¹–10¹² PFU/ml) were subjected to phenol/chloroform extraction and ethanol precipitation. Isolated phage DNA was used for restriction analysis with Eco32I, MboI, EcoRII, SalI and Csp6I restriction endonucleases (Thermo Fisher Scientific) according to supplier’s recommendations. In vitro DNA glycosylation tests were performed in the Epi Buffer using T4 phage β-glucosyltransferase (T4 BGT) and UDP-glucose from the EpiJET 5-hmC and 5-mC Analysis Kit (Thermo Fisher Scientific). DNA fragments were separated by electrophoresis in a 0.8% agarose gels stained with ethidium bromide.

Gordeeva J., Morozova N., Sierro N., Isaev A., Sinkunas T., Tsvetkova K., Matlashov M., Truncaitė L., Morgan R.D., Ivanov N.V., Siksnys V., Zeng L, & Severinov K. (2018). BREX system of Escherichia coli distinguishes self from non-self by methylation of a specific DNA site. Nucleic Acids Research, 47(1), 253-265.

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Genomic DNA Isolation and Molecular Analysis

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Standard DNA manipulations were carried out according to the protocols described by Sambrook and Russell [20 ]. Total DNA was isolated from Pseudomonas sp. ANT_H14 using a genomic DNA purification kit (Thermo Fisher Scientific, Waltham, MA, USA).
PCR reactions were performed with Phusion High Fidelity DNA polymerase (Thermo Fisher Scientific, Waltham, MA, USA). The amplified DNA fragments were analyzed by agarose gel electrophoresis and, if necessary, purified using a Gel Out kit (Thermo Fisher Scientific, Waltham, MA, USA). Subsequently, the PCR products were digested with restriction enzymes and cloned into appropriate vectors. All the constructs were confirmed by DNA sequencing. Restriction digest assay was performed in a 20-μl reaction volume under conditions recommended by the manufacturer using 0.3 μg of the phage DNA and 10 U of a restriction endonuclease (REase). The test for the presence of cohesive ends of the phage genome was performed as previously described [21 (link)], using the following REases: HindIII, SalI, EcoRI, Eco32I and PstI (Thermo Fisher Scientific, Waltham, MA, USA).

Dziewit L, & Radlinska M. (2016). Two Inducible Prophages of an Antarctic Pseudomonas sp. ANT_H14 Use the Same Capsid for Packaging Their Genomes – Characterization of a Novel Phage Helper-Satellite System. PLoS ONE, 11(7), e0158889.

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Mapping P282 Chromosomal Integration Sites

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For the determination of the chromosomal integration sites (attB) of P282, genomic DNA (gDNA) of various lysogens was isolated using the DNA mini preparation kit (Qiagen, Hilden, Germany). DNAs were digested with the restriction endonucleases Eco32I, HindIII, and XbaI according to the manufacturer’s recommendations (Thermo, St. Leon Roth, Germany). Following digestion, restriction fragments were treated with T4 ligase (Thermo) and used as template for PCR. Outward primers deduced from the 5′- and 3′-coding region of the P282 int gene were used under the following conditions: Initial PCR activation and template denaturation at 96 °C for 120 s followed by 35 cycles including denaturation phase at 96 °C for 15 s, annealing at 55 °C for 5 min and elongation for 210 s at 72 °C. In addition, a final elongation step at 72 °C for 1 min was included, before the amplicons were purified (QIAquick PCR purification kit, Qiagen) and sequenced according to Sanger (Eurofins Genomics,). The obtained nucleotide sequences of the amplicons were compared with whole genome sequences of S. aureus strains of the GenBank database (National Center for Biotechnology Information).

Kraushaar B., Hammerl J.A., Kienöl M., Heinig M.L., Sperling N., Dinh Thanh M., Reetz J., Jäckel C., Fetsch A, & Hertwig S. (2017). Acquisition of virulence factors in livestock-associated MRSA: Lysogenic conversion of CC398 strains by virulence gene-containing phages. Scientific Reports, 7, 2004.

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RT-LAMP Assay for Gene Detection

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ThermoPol Reaction Buffer Pack, AMV Reverse Transcriptase and its reaction buffer, Bst 2.0 DNA Polymerase, HiScribe™ T7 High Yield RNA Synthesis Kit, Monarch RNA Cleanup Kit, were all purchased from New England Biolabs (Beijing, China). dNTP Set 100 mM Solutions, RNase-Free DNase I and Eco32 I were obtained from Thermo Scientific (Shanghai, China). TRNzol Universal Reagent, EndoFree Mini Plasmid Kit II and Universal DNA Purification Kit were acquired from TIANGEN (Bei-jing, China). TB Green Premix Ex Taq was bought from Takara Biotechnology (Dalian, China). HiScript II Q RT SuperMix for qPCR was purchased from Vazyme (Nanjing, China). Betaine was supplied by Sigma (USA). Diethyl pyrocarbonate (DEPC) treated water, oligonucleotides and pcDNA 3.1 recombinant plasmids containing the DNA fragment of target genes were acquired from Sangon Bio-technology (Shanghai, China). The sequences of oligonucleotides are listed in Table S1. The real-time fluorescence measurements of the RT-LAMP reactions were performed using an Applied Biosystem 7500 Fast real-time PCR instrument.

Talap J., Shen M., Yu L., Zeng S, & Cai S. (2022). RT-LAMP assay combining multi-fluorescent probes for SARS-CoV-2 RNA detection and variant differentiation. Talanta, 248, 123644.

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