Pet 26b

Manufactured by Thermo Fisher Scientific

Sourced in United States

The PET-26b is a laboratory equipment from Thermo Fisher Scientific. It is designed for the analysis and characterization of materials. The core function of the PET-26b is to perform thermal analysis measurements.

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Lab products found in correlation

5 protocols using pet 26b

Cloning and Characterization of Cohnella sp. A01 Carbohydrate-Active Enzymes

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DNA extraction kits were purchased from Bioneer Company (Seoul, Korea). The restriction enzymes, NdeI, NotI, and plasmid pTZ57R/T were prepared from Fermentas (Glen Burnie, MD, USA). Plasmid extraction kit was procured from Roche Company (Switzerland). Bacterial strains including E. coli DH5α and E. coli BL21 (DE3), the expression vector pET-26b (+), and Ni-NTA resin were purchased from Invitrogen (Carlsbad, USA) and pustulan from Invivogen Company (San Diego, CA, USA). Laminarin, pullulan, starch, cellulose, and sucrose were obtained from Sigma (St. Louis, USA). Other chemicals utilized in this study were prepared from Merck (Darmstadt, Germany). Cohnella sp. A01 was obtained from shrimp pond waste water at Choebdeh (Abadan, Iran; accession No. JN208862.1)[21 (link)]. VMD 1.9 (University of Illinois, Urbana-Champaign, USA), Gene Runner 4.0 (Lynnon Biosoft, Canada), and Graphpad Prism 6 (San Diego, CA, USA) software were used to analyze protein structure and DNA sequence as well as to draw Michaelis-Menten curve and to calculate K_m and V_max. The phylogenetic tree was established using the maximum likelihood method implemented in the PhyML program (v3.1/3.0 aLRT)[22 (link)].

Rezaie M., Aminzadeh S., Heidari F., Boojar M.M, & Karkhane A.A. (2018). Biochemical Characterization of Recombinant Thermostable Cohnella sp. A01 β-Glucanase. Iranian Biomedical Journal, 22(5), 345-354.

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NoV P-based Aβ1-6 Chimeric Protein

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A novel NoV P particle-based Aβ1–6 chimeric protein without protein fusion tag was named PP-3copy-Aβ1–6-loop123-no tag and constructed as follows: A synthesized cDNA fragment encoding the NoV P domain (Hu/GII.4; GenBank: DQ078814.2) was cloned into the bacterial expression vector pET-26b (Invitrogen). Next, three copies of Aβ1–6 (DAEFRH) were inserted into loop 1, loop 2, and loop 3 of the P domain using a GGG linker. In order to improve protein expression, the NoV P domain and Aβ1–6 gene were codon-optimized. The PP-3copy-Aβ1–6-loop123 with His-tag was used as a control and was described in our previous study [36 (link), 37 (link)].

Yang P., Guo Y., Sun Y., Yu B., Zhang H., Wu J., Yu X., Wu H, & Kong W. (2019). Active immunization with norovirus P particle-based amyloid-β chimeric protein vaccine induces high titers of anti-Aβ antibodies in mice. BMC Immunology, 20, 9.

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Molecular Cloning Techniques

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DNA purification kit was obtained from PEQLAB. PCR Purification Kit was purchased from Bioneer (Seoul, Korea). Plasmid purification kit was purchased from Roche (Basel, Switzerland). TAclone PCR Cloning Kit was provided by insta-clone kit. DNA ladder, Protein molecular weight markers, Taq polymerase, Pfu DNA Polymerase, T4 DNA ligase, XhoI and NcoI enzyme, IPTG, and X-GAL were purchased from Fermentas (Glen Burnie, MD, USA). Ampicillin, kanamycin, and 2,6-dimethylphenol (DMP) were obtained from Sigma (St. Louis, USA). E. coli DH5α and E. coli BL21 (DE3) strains, and vector pET-26b (+) were obtained from Invitrogen (Carlsbad, CA, USA). Ni Sepharose resin was purchased from Qiagen. All other chemicals were provided from Merck (Darmstadt, Germany).

Shafiei M., Afzali F., Karkhane A.A., Ebrahimi S.M., Haghbeen K, & Aminzadeh S. (2019). Cohnella sp. A01 laccase: thermostable, detergent resistant, anti-environmental and industrial pollutants enzyme. Heliyon, 5(9), e02543.

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Cloning truncated human CB1 receptor

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The truncated hCB1 gene (hΔCB1, 1.1 kbp) was amplified from the hCB1 DNA template using Pfu DNA polymerase (Stratagene) and primers containing NcoI (forward) and XhoI (reverse) restriction sites in an Eppendorf Mastercycler (Westbury, NY). The PCR product was ligated into pET15b or pET26b expression vectors (Novogen) following digestion with NcoI and XhoI restriction enzymes (New England BioLabs). Colonies of One Shot Top10 E. coli cells (Invitrogen) transformed with ligation mixtures were selected on LB agar plates with antibiotic either ampicillin (Ap −100 µg/ml for pET15b) or kanamycin (Km −25 µg/ml for pET26b). After plasmid preparation (GeneJET Plasmid Miniprep Kit, Fermentas, Maryland), the presence of truncated hCB1 gene in pET15hΔCB1his6 and pET26shΔCB1his6 were confirmed by sequencing (SeqWright).

Mallipeddi S., Zvonok N, & Makriyannis A. (2018). Expression, Purification and Characterization of the Human Cannabinoid 1 Receptor. Scientific Reports, 8, 2935.

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Purification of Histidine-Tagged DdlR Protein

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A 1.48-kb PCR product containing the entire ddlR gene with six histidine codons at the 3’ end was synthesized using oBB720 and oBB721 as primers, cloned in pCR2.1-TOPO between the NdeI and BamHI sites to create pBB1852, and then recloned, using the same sites, downstream of the T7 promoter of the expression vector pET-26b (Invitrogen). The resulting plasmid, pBB1859, was introduced into E. coli strain BL21/DE3 (Studier and Moffatt, 1986 (link)), and expression of DdlR was induced in the resulting strain in L broth (at OD₆₀₀≈1.0) by adding IPTG to 0.1–0.2 mM and incubation of the cells for 1 hour (addition of IPTG caused an abrupt arrest of growth indicating DdlR toxicity to E. coli cells). DdlR-His₆ was purified to near homogeneity as described previously for B. subtilis GabR-His₆ (Belitsky, 2004 (link)). Elution from the Ni²⁺-affinity column (His·Bind resin; Novagen) was with a buffer containing 185 or 385 mM imidazole. DdlR1(K328Q)-His₆ was purified in a similar way using pBB1875. The latter plasmid is similar to pBB1859 but the ddlR1 insert was created using overlapping PCR as described above with pairs of primers M13R-oBB742 and oBB743-M13F and pBB1852 as template.

BOUILLAUT L., NEWTON W., SONENSHEIN A.L, & BELITSKY B.R. (2019). DdlR, an essential transcriptional regulator of peptidoglycan biosynthesis in Clostridioides difficile. Molecular microbiology, 112(5), 1453-1470.

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