NS1 mutants (mNS1) were generated by error-prone PCR of the pLEX-NS1 plasmid using a Gene MorphII EZ clone kit (Agilent Technology/Stratagene), according to the manufacturer’s instructions, with the following primers; mutFw 5′ AAT TCG CCC TTG AAT CGG ATC C 3′ and mutRev 5′ GGT GAT GGT GAT GAT GAC CGG T 3′. The input amount of template DNA was selected to achieve a mutation frequency between 1 nucleotide (nt) and 2nt change(s)/kilobase(kb). The E signal sequence and NS1 are 1146nt in length. Total transformed bacteria were expanded in 250ml of 2X YT broth (Fisher) containing 100μg/ml ampicillin (Sigma), plasmid DNA was purified using a Maxi prep kit (Qiagen), and packaged as lentivirus particles (pLEX-mNS1) as described previously.
Yt broth
Manufactured by Thermo Fisher Scientific
Sourced in Japan
2 × YT broth is a rich media formulation used for the growth of E. coli and other bacterial cultures in a laboratory setting. It provides a high-nutrient environment to support rapid bacterial proliferation.
Automatically generated - may contain errors
Lab products found in correlation
Ampicillin, by Merck Group (2 mentions)
Maxiprep kit, by Qiagen (1 mentions)
Lb agar, by Thermo Fisher Scientific (1 mentions)
Chloramphenicol, by Merck Group (1 mentions)
Kanamycin, by Merck Group (1 mentions)
Luria bertani agar, by Merck Group (1 mentions)
Ni sepharose, by GE Healthcare (1 mentions)
Bugbuster protein extraction reagent, by Merck Group (1 mentions)
Lb broth, by Nacalai Tesque (1 mentions)
Kao and michayluk vitamin solution, by Merck Group (1 mentions)
5 protocols using yt broth
1
Generation of lentivirus-packaged NS1 mutants
2
Culturing E. coli in 2xYT Broth
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Escherichia coli strains were cultured in 2xYT broth (Fisher Scientific) or on LB agar (Fisher Scientific) with antibiotics (Ampicillin, 50 μg ml−1) added as appropriate.
3
Competent E. coli Cultivation and Transformation
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Chemically competent TOP10 E. coli were used for DNA cloning and Gibson assembly steps. Electrocompetent TG1 E. coli were used for growth, reporter, and phage production assays. Genotypes of these E. coli strains used are listed in Supplementary Table 2 . Luria‐Bertani agar (Sigma‐Aldrich, L2897‐1KG) and M9 Minimal Media (14 g l−1 agar, 1M MgSO4, 20% (wt/vol) D‐(+)‐glucose, 1M CaCl2, 1M thiamine‐HCl) were used for cell plate growth, and 2 × YT broth (Invitrogen, 22712020) was used to culture the cells. S.O.C. medium (Formedium, SCO0202) was used to recover cells after transformation. Ampicillin (100 μg ml−1), chloramphenicol (25 μg ml−1), and kanamycin (50 μg ml−1) (Sigma‐Aldrich) were added to media where appropriate.
4
Purification of Mutant Tau Protein
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The gene encoding the human tau-441 isoform (2N4R) extended with an N-terminal His-tag and a C-terminal C-tag and containing the C291A and C322A mutations was cloned into a kanamycin resistant bacterial expression vector and transformed into BL21 (DE3) cells. A 3 ml 2-YT Broth (Invitrogen) preculture containing 25 mg/l kanamycin was inoculated from a single bacterial colony for 6 h after which it was diluted to 300 ml for overnight growth in a 1-l shaker flask and subsequently diluted to 5 -l in a 10-l wave bag. Protein expression was induced when the culture reached an OD600nm 1.0 by addition of 2 mM IPTG. Three hours after induction, the bacterial pellets were harvested by centrifugation, and lysed with Bugbuster protein extraction reagent (Merck) supplemented with a protease inhibitor cocktail (cOmplete™ ULTRA tablets EDTA free, Roche). Purification was performed by affinity chromatography using self-packed Ni-Sepharose (GE Healthcare) and C-Tag resin (Thermo Fisher Scientific) columns.
5
Recombinant Protein Expression Optimization
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BL21(DE3) was purchased from Novagen. The LB broth (Miller) was purchased from Nacalai Tesque (Japan), the M9 minimal salts premix was purchased from MP Biomedical Japan, and the 2-YT broth was purchased from Invitrogen. The glycerol minimal medium containing l- leucine (GMML)26 (link) in our study consisted of M9 minimal medium, 1 mM MgSO4, 1% glycerol, 0.3 mM l- leucine, and Kao and Michayluk vitamin solution (Sigma-Aldrich). The GMML auto-induction culture for the HV1 preparation consisted of 0.05% d- glucose, 0.2% d- lactose, 0.5% glycerol, NSP, vitamins, 10 mM O-sulfo-l -tyrosine (Watanabe Chemical Industries, Hiroshima, Japan), and carbenicillin (100 μg/ml). The auto-induction medium for the Fab preparation consisted of 2-YT, 1 mM MgSO4, 0.05% d- glucose, 0.2% d- lactose, 0.5% glycerol, nitrogen sulfur phosphorus solution (NSP), 1 mM azido-l- phenylalanine (Bachem), and kanamycin (30 μg/ml). The 20-fold concentrated NSP solution (pH6.8) was composed of 0.5 M KH2PO4 (68 g), 0.5 M Na2HPO4 (71 g), 1 M NH4Cl (53.6 g), and 0.1 M Na2SO4 (14.2 g) per liter. Anaerobic culturing was performed using Culture set, FX-2 (ISO, Japan). The optical cell densities were measured with an Ultraspec spectrophotometer (GE Healthcare) and Libra S11 Visible and UV spectrophotometers (Biochrom Ltd.).
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