Camptothecin (CPT) was provided by the Developmental Therapeutics Program (DTP), DCTD, NCI, NIH (Bethesda, MD). Human nuclear TOP1 and TOP1MT were purified from baculovirus as previously described12 (link). Plasmid pBR322 (NEB, Ipswich, MA) was used as (−) supercoiled substrates, whereas (+) supercoiled substrates were generated by incubating negative supercoiled pBR322 with Archaeoglobus fulgidus reverse gyrase following the protocol of McClendon et al.13 (link) and modified according to Hsieh and Capp29 (link) to make highly positively supercoiled DNA using an enzyme to DNA mole ratio of 40:1.
Pbr322
Manufactured by New England Biolabs
Sourced in United States
PBR322 is a commonly used bacterial plasmid vector. It contains the origin of replication and antibiotic resistance genes, allowing for propagation and selection in bacterial hosts.
Automatically generated - may contain errors
Lab products found in correlation
Puc19, by New England Biolabs (2 mentions)
Kh2po4, by Merck Group (1 mentions)
K2hpo4, by Merck Group (1 mentions)
Uv transilluminator minibispro, by DNR Bio-Imaging Systems (1 mentions)
Cy5 dctp, by GE Healthcare (1 mentions)
Qiaquick gel extraction kit, by Qiagen (1 mentions)
Qiaquick nucleotide removal kit, by Qiagen (1 mentions)
Thrombin digestion, by Merck Group (1 mentions)
E coli dh5α, by Thermo Fisher Scientific (1 mentions)
Dnase 1, by Takara Bio (1 mentions)
Pbr322, by Takara Bio (1 mentions)
Mung bean nuclease, by Takara Bio (1 mentions)
Ecori, by Takara Bio (1 mentions)
Wizard dna clean up system, by Promega (1 mentions)
Exonuclease 3, by Takara Bio (1 mentions)
Nanodrop one microvolume uv vis spectrophotometer, by Thermo Fisher Scientific (1 mentions)
E coli jm109, by Promega (1 mentions)
Ptyb21, by New England Biolabs (1 mentions)
Zymopure plasmid maxiprep kit, by Zymo Research (1 mentions)
Rnase 3, by Illumina (1 mentions)
16 protocols using pbr322
1
Preparation of Supercoiled DNA Substrates for Topoisomerase Assays
2
Plasmid DNA Topology Changes by Skyrin
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The electrophoretic monitoring of topological changes in the plasmid DNA (pBR322, New England BioLabs, Ipswich, MA, USA) induced by FeSO4 × 7H2O (Lachema, Brno, Czech republic) was used to detect the DNA-damaging and DNA-protective potential of skyrin, as [39 ,40 (link)] described in detail. In brief, the reaction mixture (final volume of 10 μL) consisted of plasmid DNA (200 ng) and either 1 mM FeSO4 × 7H2O alone (positive control) or tested concentrations of skyrin (0.0001–100 μM) alone, or combinations of skyrin with 1 mM FeSO4 × 7H2O. 0.1 M phosphate buffer (1 M KH2PO4, 1 M K2HPO4, both purchased from Sigma Aldrich, St. Louis, MO, USA; pH 7.4) was added to all samples and they were incubated 50 min in the dark at room temperature. An analysis of changes in the DNA topology caused by DNA breaks was carried out by gel electrophoresis (in 1% agarose for 90 min/100 V). The DNA was stained with GelRed dye (1 mg/mL, Sigma Aldrich, St. Louis, MO, USA) and visualized by UV illumination (UV Transilluminator MiniBISPro, DNR Bio Imaging Systems Ltd., Neve Yamin, Israel). Increases in DNA strand breakage were assayed by measuring the conversion of supercoiled DNA, form III, to relaxed circular (I) and linear forms (II). Densitometric quantification of plasmid topology forms (%) was carried out in the ImageJ 1.53c program (Wayne Rasband, National Institute of Health, Kensington, MD, USA).
3
Plasmid Copy Number Determination
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pBR322 (4,361 bp; New England Biolabs) and pUG72 (3,988 bp; originally pJJH726, EUROSCARF) were maintained in Escherichia coli and purified with a plasmid miniprep kit (GeneJet, Thermo Scientific). Each of the plasmids pBR322 and pUG72 was added post exonuclease treatment in ∼10,000 copies to each sample, 4.7E-05 ng and 4.3E-05 ng, respectively.
4
Plasmid Purification and Characterization
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All plasmids were maintained in Escherichia coli and purified with a standard plasmid miniprep kit (GeneJet, Thermo Scientific). Plasmid controls were pBR322 (4,361 bp; New England Biolabs), pUC19_yEGFP3 (3,397 bp), pUG72 (3,988 bp; originally pJJH726, EUROSCARF), pSH63 (6,998 bp), and YGPM3k20_pGP564_chrV (26305 bp; Open Biosystems).
5
Fluorescent DNA Probes for EMSA Analysis
6
Constructing a Versatile Carotenoid Biosynthesis Cassette
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The GC cassette included idsA from Archaeoglobus fulgidus DSM 4304 and crtI-crtB amplified from Pantoea agglomerans (Fig 2b ). The idsA sequence contained the geranylgeranyl diphosphate synthase (gps) gene and its constitutive promoter. Thus, the GC cassette would be constitutively expressed without induction. The products of the GC cassette were Gps, phytoene synthase (CrtB), and phytoene desaturase (CrtI), which catalyzed the generation of geranylgeranyl diphosphate (GGPP), then phytoene, and finally lycopene from endogenous IPP. The sequence of the GC cassette is given in S2 Table .
The plasmid pBRIS-GC carries the GC cassette flanked by I-SceI recognition sites. Its backbone contains pBM1 ori and the Tc resistance gene from plasmid pBR322 (New England Biolabs, Inc.). The construction process is shown inS3 Fig .
The plasmid pBRIS-GC carries the GC cassette flanked by I-SceI recognition sites. Its backbone contains pBM1 ori and the Tc resistance gene from plasmid pBR322 (New England Biolabs, Inc.). The construction process is shown in
7
Histone Octamer Purification and DNA Molecule Preparation
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Histone octamers were purchased from Abcam (ab45275) and purified by gel filtration (GE, S200). The expression plasmids yNap1 and yAsf1 were gifts from Bradley R. Carins (University of Utah) and Jessica K. Tyler (University of Texas) respectively. yNap1 (Nap1) was expressed and purified by Ni-NTA column and ion-exchange chromatography [50 (link)]. yAsf1 (Asf1) was expressed and purified by GST column and ion-exchange chromatography, and the GST tag was removed using thrombin digestion (Sigma) [51 (link)]. All the DNA molecules (211-bp, 433-bp, 614-bp and 836-bp) were prepared by PCR using pBR322 (NEB) as the template. The primer sequences are listed in Table 2 .
8
E. coli Strain Cultivation and Genetic Manipulation
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E. coli strains were grown in LB at 37 °C. Kanamycin (Kan), 50 μg/mL; tetracycline (Tet), 20 μg/mL; Spectinomycin (Sp), 80 μg/mL; Chloramphenicol (Clm), 20 μg/mL; and Ampicillin (Amp), 100 μg/mL were added to the media for selection as needed. E. coli DH5α (Life Technologies, Carlsbad, CA, USA) was the host for cloning experiments. Plasmids pEXT21 and pBR322 (NEB, Beverly; MA; USA) were used as cloning vectors. E. coli W3110 was obtained from the Coli Genetic Stock Center, Yale University, New Haven, CT, USA. Deletion of the waaL chromosomal gene in W3110 was performed according to Datsenko and Wanner as described [19 ]. Plasmids p150 [20 (link)] and p114 [21 (link)] have been described elsewhere. The different E. coli strains were from an epidemiology study from women with UTI (GlycoVaxyn AG, unpublished data). Strains, plasmids and primers used are listed in Table S1 .
9
Random Transposon Mutagenesis of A. baumannii
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A mutant library of A. baumannii was constructed by random transposon mutagenesis. A. baumannii ATCC 17978 was mutagenized using the S17–1 λ pir tra strain [32 (link)] containing pRL27, a suicide vector carrying the transposable mini-Tn5 element [33 (link)]. Tn-inserted colonies were selected by plating on LB agar plates containing 50 μg/ml kanamycin and stored at − 80 °C until use. To determine transposon insertion sites on the bacterial genome, bacterial genomic DNA was digested by BamHI. The digested DNA was ligated with BamHI-digested pBR322 (Catalogue number N3033 L, New England Biolabs, Ipswich, MA, USA) and then introduced into E. coli DH5α. The transposon insertion site was analyzed by DNA sequencing.
10
Determination of Halovirus VOLN27B Genome Type
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To determine the genome type of halovirus VOLN27B, the genome of VOLN27B was isolated from the virus stock according to the method described by Summer [23 (link)] using Wizard DNA Clean-Up System (Promega, USA). Genomic DNA (1 μg) was digested with DNase I (5 U) (TaKaRa, Japan), Exonuclease III (20 U) (TaKaRa, Japan), and Mung Bean Nuclease (4 U) (TaKaRa, Japan) at 37°C for 1 h, and the resulting product was checked by DNA electrophoresis. An equal amount of undigested plasmid pBR322 (NEB, USA) was used as a circular dsDNA standard, and the EcoRI (TaKaRa, Japan) cleaved pBR322 was used as a linear dsDNA standard. These untreated and EcoRI leaved pBR322 plasmids were also used as controls in treatments nucleases DNase I, Exonuclease III, and Mung Bean Nuclease.
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