Chemicals were purchased from Sigma-Millipore Inc (St Louis, MO), Research Products International Inc (Mount Prospect, IL), and Gold Biotechnology Inc (St Louis, MO). Oligonucleotides for cloning were purchased from Integrated DNA Technologies (Coralville, IA). Enzymes for molecular biology were purchased from New England Biolabs (Ipswich, MA). All reagents and buffers were thoroughly degassed using alternating cycles of vacuum and nitrogen pressure on a home-built Schlenk line apparatus. Anaerobic conditions were maintained via airtight syringes, excess reductant (dithionite), and a vinyl glove box (Coy Laboratories, MI) under a nitrogen (95%) and hydrogen (5%) mix atmosphere.
Enzymes for molecular biology
Manufactured by New England Biolabs
Sourced in Germany
Enzymes for molecular biology are essential tools used in various laboratory procedures. They catalyze specific chemical reactions, enabling researchers to manipulate and analyze DNA, RNA, and proteins. These enzymes play a crucial role in techniques like cloning, sequencing, and genetic engineering.
Automatically generated - may contain errors
Lab products found in correlation
Oligonucleotides for cloning, by Integrated DNA Technologies (1 mentions)
Q sepharose, by GE Healthcare (1 mentions)
Heparin, by GE Healthcare (1 mentions)
S200 size exclusion chromatography, by GE Healthcare (1 mentions)
Biogel p4 resin, by Bio-Rad (1 mentions)
Bl21 ai cells, by Thermo Fisher Scientific (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Oligonucleotide, by Integrated DNA Technologies (1 mentions)
Nucleospin plasmid purification kit, by Macherey-Nagel (1 mentions)
Gel extraction kit, by Qiagen (1 mentions)
Expand high fidelity polymerase, by Roche (1 mentions)
Vent polymerase, by New England Biolabs (1 mentions)
Trio thermoblock, by Analytik Jena (1 mentions)
Molecular weight marker, by Thermo Fisher Scientific (1 mentions)
Size exclusion column, by GE Healthcare (1 mentions)
Nitrocellulose membrane, by GE Healthcare (1 mentions)
Pyes2 ct vector, by Thermo Fisher Scientific (1 mentions)
Anti v5 hrp, by Thermo Fisher Scientific (1 mentions)
Glass econocolumns, by Bio-Rad (1 mentions)
Histrap column, by GE Healthcare (1 mentions)
12 protocols using enzymes for molecular biology
1
Anaerobic Molecular Biology Procedures
2
Protein Purification Using Chromatography
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Standard laboratory chemicals and protease inhibitor cocktail (PIC) were purchased from Sigma-Aldrich (St. Louis, MO, USA) and Research Products International (Mt. Prospect, IL, USA). Q-sepharose, Heparin and S200 size exclusion chromatography resins were from GE Healthcare (Pittsburgh, PA, USA). Ni2+-NTA agarose was from Gold Biotechnology (St. Louis, MO). Biogel-P4 resin was from Bio-Rad Laboratories (Hercules, CA, USA). Enzymes for molecular Biology were from New England Biolabs (Ipswich, MA, USA). Oligonucleotides were purchased from Integrated DNA Technologies (Coralville, IA). Fmoc-4-amino-phenylalanine was from Angene International Ltd. (Nanjing, China). Commercial 4-azidophenylalanine was purchased from Chem-Impex International (Wood Dale, IL, USA). MB543 DBCO was purchased from Click Chemistry Tools (Scottsdale, AZ, USA). Alexa Fluor 594 DIBO alkyne and BL21Ai cells were purchased from ThermoFisher Scientific (Waltham, MA, USA).
3
Molecular Cloning Protocols and Plasmid Construction
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Oligonucleotides used for plasmids construction and information about the construction strategies are available upon request. DNA manipulations were performed as described (Sambrook et al., 1989 ), or with the Getaway cloning system (Life Technologies) in the case of lentiviral vectors. Enzymes for molecular biology were obtained from New England Biolabs. Plasmids were purified with the Nucleospin plasmid purification kit (Macherey-Nagel 740422.10). Linear DNA was purified from agarose gels using the gel extraction kit from Qiagen. Polymerase chain reactions (PCRs) were performed with the Expand High Fidelity polymerase (Roche) and a TRIO-thermoblock (Biometra GmbH). Plasmids used are listed in Table 1 . E. coli DH5α (Chan et al., 2013 (link)) was used to amplify plasmids. All plasmids generated in this work are available for non-commercial purposes under request.
4
DNA Manipulation and Plasmid Construction
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DNA manipulations were performed as described previously (Sambrook et al., 1989 ). Enzymes for molecular biology were obtained from New England Biolabs. PCRs were performed with a Vent polymerase (New England Biolabs) and a TRIO thermoblock (Biometra). All plasmids used in this study bear ampicillin resistance for selection in Escherichia coli and are listed in Table 2 , where the yeast features and inserts are described. The osh2-HHR* ORF was synthesized by Genescript. Information about the construction strategies are available upon request. The oligonucleotides used in this work for plasmid construction are listed below (Table 3 ).
5
Molecular Biology Enzyme Purification
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Enzymes for molecular biology were from New England Biolabs. TOP10 ultracompetent cells, the pYES2CT vector, anti-V5-HRP, molecular weight markers and native PAGE gels and reagents were from Life Technologies. Glass econocolumns and the DC protein assay kit were from BioRad. Denatured sheared salmon sperm DNA and reagents for assays of esterase and acyltransferase activity were from Sigma, except p-nitrophenyl hexanoate from Ark chemicals. HisTrap columns, PD-25 gel filtration columns, size-exclusion columns, protein chromatography standards and nitrocellulose membrane were from GE Healthcare. Precast acrylamide gels were from NuSep. LumiGLO chemiluminescence reagents were from Cell Signaling Technology.
6
Heterologous Expression of Kv1.2 and hERG
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pPIC3.5-rat Kv1.2 containing the His9 tag at the N-terminus7 and hERG cDNA in pSP64 were kindly provided Dr. D. Parcej and Prof. S.A. Goldstein respectively. KM71 Pichia pastoris cells were from Invitrogen. Enzymes for molecular biology were purchased from New England Biolabs, Promega or Stratagene. All general chemicals were obtained from Sigma Chemicals Co. Mouse anti-His tag, goat anti-mouse HRP were purchased from Novagen and BioRad laboratories respectively.
7
Chemical Reagents and Enzymes Sourcing
8
Molecular Biology Reagent Sourcing
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All chemicals were purchased from ROTH (Karlsruhe, Germany) and Merck (Sigma-Aldrich, Burlington, MA, US), unless stated otherwise. Enzymes for molecular biology were purchased from New England Biolabs GmbH (Frankfurt am Main, Germany).
9
Routine Molecular Biology Techniques in Microbiology
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Routine molecular biology techniques were performed as described before37 . PCR primers used in this study were purchased from Integrated DNA Technologies (IDT) (Belgium) (Table 2 ). Enzymes for molecular biology were purchased from New England Biolabs and used according to the suppliers’ instructions. Plasmid DNA from E. coli was purified using QIAGEN miniprep kits. Chromosomal DNA from L. rhamnosus GR-1 was isolated as previously described19 (link).
10
Chemical Reagent Procurement Protocol
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All chemicals were purchased from ROTH (Karlsruhe, Germany) and Merck (Sigma-Aldrich, Missouri, USA), unless stated otherwise. Enzymes for molecular biology were purchased from New England Biolabs GmbH (Frankfurt am Main, Germany).
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