All chemicals were of analytical grade and were purchased from Sigma-Aldrich (USA), Qualigens (India), Merck (India), Himedia (India) and SRL (India). LB media was purchased from Himedia (India). Ni+2-NTA resin, Protein G beads and Taq polymerase were purchased from Merck-Millipore (USA). cDNA synthesis kit was purchased from Thermo-Fisher (USA). Biorex-70 resin was purchased from Bio-Rad (USA). Cell culture chemicals were purchased from Sigma-Aldrich (USA) while cell culture plasticware was from Corning (USA). SYBR Green mix was purchased from Kapa Biosystems (USA).
Lb media
Manufactured by HiMedia
Sourced in India
LB media is a commonly used nutrient-rich growth medium for the cultivation of bacteria in laboratory settings. It provides the necessary nutrients and conditions for the optimal growth and propagation of a wide range of bacterial species. The core function of LB media is to support the proliferation of bacterial cultures, enabling researchers to study and manipulate these microorganisms for various scientific and experimental purposes.
Automatically generated - may contain errors
Lab products found in correlation
Ni2 nta resin, by Merck Group (1 mentions)
Cdna synthesis kit, by Thermo Fisher Scientific (1 mentions)
Sybr green mix, by Roche (1 mentions)
Protein g beads, by Merck Group (1 mentions)
Taq polymerase, by Merck Group (1 mentions)
Cell culture plasticware, by Corning (1 mentions)
Biorex 70 resin, by Bio-Rad (1 mentions)
Cell culture chemicals, by Merck Group (1 mentions)
Isopropyl β d 1 thiogalactopyranoside (iptg), by Bio Basic (1 mentions)
Ampicillin, by Bio Basic (1 mentions)
Ampicillin, by Merck Group (1 mentions)
Restriction enzyme, by New England Biolabs (1 mentions)
Kanamycin, by Merck Group (1 mentions)
Ninhydrin, by Merck Group (1 mentions)
Tetrahydro 2 furoic acid, by Merck Group (1 mentions)
Dna ligation kit, by New England Biolabs (1 mentions)
Lsm 780, by Zeiss (1 mentions)
Lsm image browser software, by Zeiss (1 mentions)
Sodium chloride (nacl), by HiMedia (1 mentions)
Yeast extract, by HiMedia (1 mentions)
5 protocols using lb media
1
Protein Purification and Characterization
2
Purification of Optineurin Protein
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The optineurin clone pDEST17-hOPTN was a gift from Jon Ashwell (Addgene plasmid #23053; http://n2t.net/addgene : 23053; RRID: Addgene_23053). The clone was transformed into the BL21-Gold (DE3) E. coli expression host. Cells were grown in LB media (HiMedia, India) containing 100 mM ampicillin (Biobasic Inc.) in an incubator shaker (New Brunswick™ Innova® 44/44R) at 37 °C till an OD600 of 0.6, following induction with 1 mM IPTG (Biobasic Inc.) cells were allowed to grow further for 16 hours, before harvesting. The protein expression was checked by boiling small culture aliquots in an SDS-loading buffer and running on 12% SDS-PAGE. The cells were harvested by centrifugation. The pellet was sonicated in lysis buffer (50 mM sodium phosphate, 300 mM NaCl, and 10 mM imidazole, pH 8.0). The 6× His-tagged OPTN was purified under native conditions using Ni-NTA affinity chromatography. Washing and elution were done with imidazole concentrations ranging from 50–500 mM. Eluted fractions showing OPTN band on SDS-PAGE were pooled and dialyzed against 50 mM sodium phosphate and 50 mM NaCl, pH 8.0. Dialysed sample was concentrated using a 30 kDa cut-off centricon and stored at −80 °C freezer until further use.
3
Plasmid Maintenance and Culture
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Plasmids were maintained
in E. coli DH5α33 (link) and were cultured at 37 °C in LB media (Himedia, USA),
which includes yeast extract (0.5%) along with tryptone (1%) and NaCl
(0.5%) with antibiotic ampicillin (Merck, USA) (100 mg/mL) as a selection
marker. Pseudomonas sp. K-62 was a
gift from Masako Kiyono (Tokyo, Japan). The plasmid containing E. coli strain was grown at 25 °C in the defined
media of tryptic soy broth. Subculturing was done by adding the primary
culture from the log phase having an optical density (OD) of 0.4.
in E. coli DH5α33 (link) and were cultured at 37 °C in LB media (Himedia, USA),
which includes yeast extract (0.5%) along with tryptone (1%) and NaCl
(0.5%) with antibiotic ampicillin (Merck, USA) (100 mg/mL) as a selection
marker. Pseudomonas sp. K-62 was a
gift from Masako Kiyono (Tokyo, Japan). The plasmid containing E. coli strain was grown at 25 °C in the defined
media of tryptic soy broth. Subculturing was done by adding the primary
culture from the log phase having an optical density (OD) of 0.4.
4
Molecular Biology Reagents Protocol
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Antibiotics (kanamycin and gentamycin), ninhydrin, tetrahydro-2-furoic acid (THFA) and 2′, 7′-dichlorohydrofluorescein-diaceate (H2DCFDA) were purchased from Sigma (St. Louis, MO). Proline was purchased from SRL (India). Sulphosalicylic acid, LB media and salts were purchased from Himedia, India. Restriction enzymes and DNA ligation kit were purchased from New England Biolabs (NEB, USA) and Fermentas (USA) respectively. Pfu polymerase was purchased from Stratagene (Agilent, USA).
5
Effect of AgNPs on E. coli Cytoskeleton
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LB media, agar-agar, yeast extract, trypton and NaCl, required for microbial culture were purchased from HiMedia Laboratories (Mumbai, India). Petri plates were obtained from Tarsons Products Pvt. Ltd (Kolkata, India). The E. coli strain expressing FtsZ-GFP (WM2724) was a kind gift from Dr. William Margolin (University of Texas Medical School, USA)26 (link). The construct expressing mCherry-FtsA (pSZ50 BsFtsA) was a kind gift from Dr. Jan Löwe (MRC Laboratory of Molecular Biology, Cambridge, UK)27 (link). BsFtsA construct was stably integrated into E. coli strain DH5α. Both E. coli strains were cultured in LB media and induced with 50 μM IPTG for 2H before adding AgNPs at the indicated concentrations (50 μM or 125 μM). The AgNP treated and untreated controls were incubated in shaking incubator (220 RPM, 37 °C) and live bacteria were immobilized on a pad of LB containing 1.5% low-melting-point agarose and covered with glass cover slips. The green and red fluorescence of the cytoskeletal proteins were detected using confocal microscope (LSM780, Carl-Zeiss, Germany) and the images were analyzed using LSM Image Browser software (Zeiss, Germany).
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