DUOX1 (AF542180.1) coding sequences (CDS) were synthesized and validated, and then inserted into pcDNA3.1(+) vector (Addgene, USA) at Hind III/EcoR I sites. DUOX1 overexpression plasmids (oeDUOX1) were extracted using a Plasmid Extraction Kit (Solarbio, Beijing). The sequences of DUOX1 siRNA (siDUOX1) at 3 different sites were synthesized and listed in Table 1 .
Plasmid extraction kit
Manufactured by Solarbio
Sourced in China
The Plasmid Extraction Kit is a laboratory tool designed to isolate and purify plasmid DNA from bacterial cultures. It utilizes a standard alkaline lysis method to extract the plasmid DNA, which can then be used for various downstream applications, such as cloning, sequencing, or transfection.
Automatically generated - may contain errors
Lab products found in correlation
Pet28a, by Thermo Fisher Scientific (2 mentions)
Dna purification kit, by Solarbio (2 mentions)
T4 dna ligase, by Thermo Fisher Scientific (2 mentions)
Restriction endonuclease, by Thermo Fisher Scientific (2 mentions)
Phusion dna polymerase, by Thermo Fisher Scientific (2 mentions)
Pcr product purification kit, by Solarbio (2 mentions)
Pcdna3.1 vector, by Addgene (1 mentions)
Xylose x1, by Merck Group (1 mentions)
Alkaline phosphatase, by Thermo Fisher Scientific (1 mentions)
Trans cinnamic acid, by Merck Group (1 mentions)
Ecori, by Takara Bio (1 mentions)
Bca protein assay kit, by Solarbio (1 mentions)
P coumaric acid, by Merck Group (1 mentions)
Hindiii, by Takara Bio (1 mentions)
3 full race core set, by Takara Bio (1 mentions)
Isopropyl β d thiogalactopyranoside, by Solarbio (1 mentions)
4 nitrophenyl β d xylopyranoside pnpx, by Merck Group (1 mentions)
T4 dna ligase, by Takara Bio (1 mentions)
Restriction endonuclease, by Takara Bio (1 mentions)
Genemorph 2 random mutagenesis kit, by Agilent Technologies (1 mentions)
5 protocols using plasmid extraction kit
1
DUOX1 Overexpression and Silencing Protocol
2
Construction and Characterization of Recombinant Xylanases
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The recombinant strains pET-XynA and pET-XynA-Tr were constructed previously and used as the template in this study [10 (link)]. The expression vector pET-28a(+) was obtained from Invitrogen (Carlsbad, CA, USA). The stains of E. coli DH5α and BL21 (DE3) were cultured in Luria-Bertani (LB) medium (1% w/v tryptone, 0.5% w/v yeast extract, and 1% w/v NaCl) at 37 °C. Phusion DNA polymerase, restriction endonuclease, T4 DNA ligase, and alkaline phosphatase were purchased from ThermoFisher Scientific (Shanghai, China). A plasmid extraction kit and DNA purification kit were bought from Solarbio (Beijing, China). Xylan from beechwood used as xylanase substrate, xylooligosaccharide standards including xylobiose (X2), xylotriose (X3), xylotetraose (X4), xylopentaose (X5), and xylohexaose (X6) as well as xylose (X1) were all obtained from Sigma (St. Louis, MO, USA). All other analytical grade chemicals were commercially available.
3
Rhodotorula glutinis Cinnamic Acid Biosynthesis
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The strains used in this work included Rhodotorula glutinis CGMCC2258 and E. coli BL21 (DE3), Rosetta-Gami 2 (DE3), and DH5α. The plasmids PMD-18T and pET-30a were used for gene cloning and expression, respectively. TransStart FastPfu DNA polymerase DNA kit, Plasmid Extraction kit, HisPur™ Ni-NTA Resin, and BCA Protein Assay kit were purchased from Beijing Solarbio Science & Technology Co., Ltd (Beijing, China). The restriction endonucleases (HindIII and EcoRI) and the rapid amplification of cDNA end (RACE) kits (3′-Full RACE Core Set and 5′-Full RACE) were purchased from Takara (Shiga, Japan). Yeast extract and peptone were purchased from Oxoid (Basingstoke, Hampshire, UK). All solvents for high-performance liquid chromatography (HPLC) analysis were purchased from Fisher Scientific (Fair Lawn, NJ, USA). All chemicals, including standards of trans-cinnamic acid and p-coumaric acid, were purchased from Sigma–Aldrich (St. Louis, MO, USA) unless indicated otherwise.
4
Random Mutagenesis of Recombinant Xylanase
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The recombinant strain pET28a(+)-Xyl21 was constructed previously [11 (link)] and used as the template in this study. A GeneMorph II Random Mutagenesis Kit used for the construction of the mutant library was purchased from Agilent Technologies (California, USA). Restriction endonucleases, pfu DNA polymerases, alkaline phosphatases, and T4 DNA ligases were purchased from TaKaRa (Dalian, China). Escherichia coli BL21 (DE3) was used for protein expression. The recombinant strains were cultured at 37 °C in Luria-Bertani (LB) medium (1% w/v NaCl, 0.5% w/v yeast extract, and 1% w/v tryptone). The Plasmid Extraction Kit, DNA Purification Kit, PCR Product Purification Kit, and isopropyl-β-D-thioga-lactopyranoside (IPTG) were purchased from Solarbio (Beijing, China). The substrate 4-nitrophenyl β-D-xylopyranoside (pNPX) was acquired from Sigma (St. Louis, MO, USA). Phosphate buffer (10 mM) contained 8 g/L NaCl, 3.58 g/L Na2HPO4·12H2O, 0.2 g/L KCl, 0.27 g/L KH2PO4. The rest of the chemicals were of analytical grade and commercially available.
5
Recombinant Protein Expression in E. coli
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The plasmid pET-28a from Invitrogen (Carlsbad, CA, USA) was adopted as the expression vector in Escherichia coli DH5α and BL21 (DE3) were utilized to clone and expression of hosts, separately. The recombinant strains of E. coli were cultivated in LB medium (0.5% w/v yeast extract, 1% w/v tryptone, and 1% w/v NaCl). Phusion DNA polymerase, restriction endonuclease, and T4 DNA ligase were purchased from ThermoFisher Scientific (Shanghai, China). The plasmid extraction kit, PCR product purification kit, and isopropyl-β-D-thioga-lactopyranoside (IPTG) were purchased from Solarbio (Beijing, China). Nickel columns and nickel resin used for affinity chromatography were purchased from GE Health-care (Uppsala, Sweden). Substrate FB1 (purity of 98%) was obtained from Pribolab Ltd. (Qingdao, China, http://wwwpribolab.com/ , accessed on 4 March 2022) and dissolved in acetonitrile-water. All additional reagents and chemicals are of analytical grade.
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