All cloning steps were performed in E. coli NovaBlue cells (Novagen). Expression was conducted in E. coli BL21(DE3)-CodonPlus-RIL or C41(DE3) cells (Stratagene). Sources of supplies were as follows: pET vectors from Novagen; Pfu Turbo polymerase from Stratagene; T4 DNA ligase and restriction enzymes from New England Biolabs; plasmid extraction kits from Sigma; Terrific Broth medium from Difco; thrombin (T-3399) and acyl-CoAs from Sigma; NADH and NADPH from Roche Diagnostics; Calbiosorb resin from Calbiochem; Ni-NTA agarose from Qiagen; HiTrap Q XL and HisTrap HP columns from Amersham Biosciences; Econo-Pac columns from BioRad. All other supplies were reagent grade or better. An AEKTA FPLC (Amersham Biosciences) was used for protein purification. Spectrophotometric measurements were carried out on a Cary 50 conc Varian Spectrophotometer. Solutions were shaken using a Stuart see-saw rocker (SSL-4). Proteins were analyzed by conformational sensitive gel electrophoresis (15% CS-PAGE) [34] (link), [35] (link), [36] (link) using gels routinely containing 5 M urea and polymerized overnight, standard 10% SDS-PAGE [37] (link) and 10% Tris-Tricine SDS-PAGE [38] (link). Identity and homogeneity of acyl-PfACPs was confirmed by MALDI-TOF-MS. Protein concentrations were measured using the Bradford assay and BSA as a standard [39] (link).
Plasmid extraction kit
Manufactured by Merck Group
Sourced in United States
The Plasmid Extraction Kit is a laboratory tool designed to isolate and purify plasmid DNA from bacterial cultures. It utilizes a series of buffers and centrifugation steps to extract and concentrate the plasmid DNA, enabling its use in downstream applications such as cloning, sequencing, and transfection.
Automatically generated - may contain errors
Lab products found in correlation
T4 dna ligase and restriction enzymes, by New England Biolabs (1 mentions)
Ni nta agarose, by Qiagen (1 mentions)
Econo pac column, by Bio-Rad (1 mentions)
Hitrap, by Cytiva (1 mentions)
Mda mb 231, by American Type Culture Collection (1 mentions)
Transwell plate, by BD (1 mentions)
Mcf 7, by American Type Culture Collection (1 mentions)
Rt pcr reagents, by Merck Group (1 mentions)
Rock inhibitor, by Santa Cruz Biotechnology (1 mentions)
Dna purification kit, by Merck Group (1 mentions)
Effectene transfection kit, by Qiagen (1 mentions)
E coli jm109 competent cells, by Takara Bio (1 mentions)
Pmdtm19 t vector, by Takara Bio (1 mentions)
Escherichia coli dh5α competent cells, by Takara Bio (1 mentions)
6 protocols using plasmid extraction kit
1
Cloning and Expression of Acyl-PfACPs
2
Investigation of FAP-α and FAK in Breast Cancer
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Human breast cancer cells, MCF7 and MDA-MB-231 were from the ATCC (American Type Cell Collection, Manassas, VA, USA). Fresh frozen human breast tissues were collected from University Hospital of Wales under the approval of the local ethical committee, obtained immediately after surgery and stored at -80°C until used.
Antibodies to human FAP-α (sc-100528 and ab5066), FAK (sc-1680) and pFAK (sc-11765-R were from Santa-Cruz Biotechnologies, Inc. (Santa Cruz, CA, USA or Abcam, Cambridge, UK). ROCK inhibitor was from Santa-Cruz Biotechnologies, Inc. (Santa Cruz, CA, USA), ERK inhibitor, Wortmannin, and Wiskostatin were from Calbiochem (Nottingham, UK). Matrigel (reconstituted basement membrane) was purchased from Collaborative Research Products (Bedford, MA, USA). Transwell plates equipped with a porous insert (pore size 8 μm) were from Becton Dickinson Labware (Oxford, UK). RT-PCR reagents and plasmid extraction kits were from Sigma (St. Louis, MO, USA).
Antibodies to human FAP-α (sc-100528 and ab5066), FAK (sc-1680) and pFAK (sc-11765-R were from Santa-Cruz Biotechnologies, Inc. (Santa Cruz, CA, USA or Abcam, Cambridge, UK). ROCK inhibitor was from Santa-Cruz Biotechnologies, Inc. (Santa Cruz, CA, USA), ERK inhibitor, Wortmannin, and Wiskostatin were from Calbiochem (Nottingham, UK). Matrigel (reconstituted basement membrane) was purchased from Collaborative Research Products (Bedford, MA, USA). Transwell plates equipped with a porous insert (pore size 8 μm) were from Becton Dickinson Labware (Oxford, UK). RT-PCR reagents and plasmid extraction kits were from Sigma (St. Louis, MO, USA).
3
Ellagic Acid Cell Assay Protocol
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Ellagic acid was purchased from Dalian Meilun Biological Technology Co., Ltd., with a mass fraction of 99%. Fetal bovine serum, DMEM glucose medium, and trypsin were purchased from the HyClone Company (USA). The DNA purification kit and Plasmid Extraction kit were purchased from Sigma Company (Germany). The Effectene transfection kit was purchased from Qiagen Company (USA), and the rabbit anti-human IGFBP7 was purchased from Abcam Corporation (UK). The RT-PCR kit and cDNA reverse transcription kit was purchased from Japan Toyobo Corporation (Japan).
4
16S rRNA Gene Clone Library Construction
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DNA samples from ANSBR 1 on d 91 were used to construct a 16S rRNA gene clone library. 16S rRNA gene fragments from the purified DNA were PCR-amplified using a PCR primer set of 27f and 1492r as described above. The PCR products were purified using the Qbiogene Geneclean Spin kit (MP Biomedicals, Santa Ana, CA, USA) and subcloned using the pTBlue Perfectly Blunt cloning kit (Novagen, Madison, WI, USA) and E. coli JM109 competent cells (Takara). Plasmid DNA was purified using a plasmid extraction kit (Sigma-Aldrich, St. Louis, MO, USA) according to the manufacturer’s instructions and subjected to the analyses described below.
5
Plasmid Extraction and Quantification
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Mycobacterial DNA was prepared as described previously by Douarre et al. (2010 (link)). Plasmid DNA was isolated from E. coli DH5α using a plasmid extraction kit as per the manufacturer's instructions (Sigma Aldrich). Plasmid isolation from Lactobacillus strains was also carried out using this kit, after initial incubation (30 min at 37°C) in protoplast buffer (20 mM Tris-HCl, 5 mM EDTA, 0.75 M Sucrose, 10 mg/ml lyzozyme and 50 U/ml mutanolysin pH 7.5). Quantitative analysis was carried out using microspectrophotometry (Nanodrop, De, USA) and plasmid DNA concentration was normalized to 250 ng/μl.
6
Plasmid Extraction and Cloning
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All chemicals and reagents were purchased from Sigma-Aldrich (St. Louis, MO, United States), unless otherwise indicated.
DNA Marker 100/2000 (3422A; TaKaRa), Escherichia coli DH5α Competent cells (9057; TaKaRa), Plasmid extraction kit (PLN350; Sigma-Aldrich), T-Vector pMDTM 19 (3271; TaKaRa).
DNA Marker 100/2000 (3422A; TaKaRa), Escherichia coli DH5α Competent cells (9057; TaKaRa), Plasmid extraction kit (PLN350; Sigma-Aldrich), T-Vector pMDTM 19 (3271; TaKaRa).
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