UA (CAS 6159-44-0) was obtained from Spectrum Chemical Mfg. Corp. (Gardena, CA, USA) and used as received. The 234U/238U atom ratio was 7.6 ± 2 × 10−6, consistent with the depleted form of uranium (Wilson et al., 2014 (link)). Cis-diamminedichloro-platinum (II) (CAS 15663-27-1, cisplatin) was obtained from Selleck Chemicals (Houston, TX, USA); potassium dichromate (CAS 7778-50-9) was obtained from Mallinckrodt Baker, Inc. (Paris, KY, USA) and sodium (meta)arsenite (CAS 7784-46-5) was obtained from Aldrich Chemical Co. (Milwaukee, WI, USA). All were used as received. N-(2-acetamido)-2-aminoethanesulfonic acid (ACES) buffer (VWR International, Radnor, PA, USA), was prepared as 33.0 mM or 50.0 mM stock solutions, and the desired pH was obtained by addition of NaOH or HCl at 37 °C. Buffers were treated with Chelex® 100 resin (Bio-Rad Laboratories, Hercules, CA, USA) followed by 0.2 μm filtration to remove trace metals. All metal solutions were prepared immediately before use. The final pH values of reaction solutions were measured in mock 2 ml reaction solutions. Reactions with UA in pH 7.44 ACES buffer yielded a final reaction pH 7.40 ± 0.05 at 37 °C. The pBR322 plasmid was obtained from New England Biolabs (Ipswich, MA, USA) and diluted in water and reacted at final concentrations of 0.30 ± 0.08 mM DNA-P.
Pbr322 plasmid
Manufactured by New England Biolabs
Sourced in United States
The PBR322 plasmid is a commonly used cloning vector. It is a circular, double-stranded DNA molecule that can be used to propagate and maintain genes of interest in bacterial cells. The PBR322 plasmid contains an origin of replication, allowing it to replicate independently within the host cell, and a selectable marker gene, which enables the identification and selection of cells containing the plasmid.
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Lab products found in correlation
8 protocols using pbr322 plasmid
1
Depleted Uranium Oxidation Kinetics
2
pDNA Cleavage Assay with pBR322 Plasmid
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pDNA cleavage assay with the use of pBR322 plasmid (New England BioLabs Inc., N3033L) was performed as reported previously [5 (link), 36 (link)]. In this assay, all samples contained 0.2 μg pDNA in sodium phosphate buffer (25 mmol L−1 sodium phosphate, 50 μmol L−1 DTPA, pH 7.4, 37°C). After addition of compounds, the resulting mixtures were incubated for 30 min at 37°C. All concentrations listed in the section were final in the samples. After incubation, the reaction mixtures were subjected to 0.6% agarose gel electrophoresis. Samples were electrophoresed in TBE buffer (89 mmol L−1 Tris, 89 mmol L−1 boric acid, and 2 mmol L−1 EDTA) at 5.5 V cm−1 for 2 h; gels were stained with GelRed™ Nucleic Acid Gel Stain and photographed using a UV transilluminator. Integrated densities of all pBR322 forms in each lane were quantified using the TotalLab TL100 image analysis software to estimate pDNA cleavage efficiency (Nonlinear Dynamic Ltd., USA).
3
Hairpin Construct Synthesis for Helicase Studies
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The hairpin construct was synthesized28 (link),35 (link) by ligating a variable 89-bp hairpin stem capped by a (dT)4 tetraloop to two 1.5-kb double-stranded handles made by PCR amplification of sections of the pBR322 plasmid (New England Biolabs, Ipswitch, MA, USA). The left and right handles were respectively modified with 5′ biotin and digoxigenin to facilitate attachment to streptavidin- and anti-digoxigenin antibody-coated beads. In the final construct, the hairpin insert was flanked on the 3′ side by a poly-dT loading site for helicase binding (Fig. 1b ). For experiments probing the stepping behavior of monomeric UvrD, the loading site was 10 nt long to ensure that only a monomer of UvrD bound to the DNA. A longer loading site of 19, 38, or 60 nt was used for experiments examining the stepping dynamics of dimeric UvrD. All oligonucleotides were purchased from Integrated DNA Technologies (Coralville, IA, USA). Sequences for all hairpin inserts and primers are displayed in Supplementary Table 1 .
4
Production of DMOS DNA Registers
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The DMOS registers are made up of DNA sequences that have at least two dTdCdR sites (R = Purine, A or G). These sites have been proven to be effective binding sites based on the research conducted by Chari24 (link) and synthesized by TWIST Bioscience. To facilitate the mass production of DMOS registers, we assembled the registers into the pBR322 plasmid (New England Biolabs) using the NEBuilder© HiFi Assembly Master Mix. We inserted the register into the plasmid by cleaving the pBR322 plasmid using FastDigest restriction enzymes Bsu15I and EcoRI.
Next, we transformed the modified plasmid into NEB 5-ɑ competent Escherichia coli bacteria and grew them under Carbenicillin antibiotic resistance. To extract the assembled plasmid, we used the Monarch Plasmid Miniprep Kit (NEB).
Next, we transformed the modified plasmid into NEB 5-ɑ competent Escherichia coli bacteria and grew them under Carbenicillin antibiotic resistance. To extract the assembled plasmid, we used the Monarch Plasmid Miniprep Kit (NEB).
5
Plasmid DNA Cleavage Assay
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The pBR322 plasmid (N3033L, New England BioLabs Inc., Frankfurt, Germany) was used in pDNA cleavage assay that was performed according to our previous report [8 (link)]. In this assay, all samples contained 0.2 µg pDNA in a sodium phosphate buffer (25 mM sodium phosphate, 50 µM DTPA, pH 7.4, 37 °C). After addition of compounds, the resulting mixtures were incubated for 30 min at 37 °C. Afterwards, the reaction mixtures were subjected to 0.6% agarose gel electrophoresis. The samples were electrophoresed in TBE buffer (89 mM Tris, 89 mM boric acid, 2 mM EDTA, pH 8.0) at 5.5 V/cm for 2 h. Gels were stained with Gel Red™Nucleic Acid Gel Stain and photographed using the Odyssey Fc Imaging System (LI-COR Biotechnology, Bad Homburg, Germany). The integrated densities of two identified pBR322 forms (supercoiled and nicked circular form) in each lane were quantified using Image Studio analysis software (LI-COR Biotechnology, Bad Homburg, Germany) to estimate pDNA cleavage efficiency.
6
Plasmid DNA Cleavage Assay
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A pDNA cleavage assay with the use of pBR322 plasmid (New England BioLabs, Inc., N3033 L, Ipswich, MA, USA) was performed as reported previously [13 (link)]. In this assay, all samples contained 0.2 μg pDNA in the sodium phosphate buffer (25 mmol L−1 sodium phosphate, 50 μmol L−1 DTPA, pH 7.4). FeCl2 (150 µmol L−1) was used in control experiments. Powdered KO2 was dissolved by 4 mmol L−1 BMPO in the buffer containing 10% DMSO (v/v), vortexed for 10 s and 10 µL of the mixture was added into 10 µL solution of pDNA. The resulting mixtures were incubated for 30 min at 37 °C. After incubation, the reaction mixtures were subjected to 0.6% agarose gel electrophoresis. Integrated densities of all pBR322 forms in each lane were quantified using the Image Studio analysis software (LI-COR Biotechnology, Bad Homburg, Germany) to estimate pDNA-cleavage efficiency.
7
pDNA Cleavage Assay with ASRE
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A pDNA cleavage assay with the use of pBR322 plasmid (New England BioLabs, Inc., N3033 L, Ipswich, MA, USA) was performed as reported previously [31 (link)]. In this assay, all samples contained 0.2 μg pDNA in the final sodium phosphate buffer (25 mmol L−1 sodium phosphate, 50 μmol L−1 DTPA, pH 7.4). Five microliters of the Na2S, Na2Sn, Cys or GSH aqueous stock solution (in final 100 μmol L−1) was added to 15 µL of pDNA solution containing ASRE (in mg mL−1: 0, 0.23, 0.47, 0.94, 1.41 or 1.88) or its chemical component (in mmol L−1: 0.2, 0.4, 0.8, 1.2 or 1.6). All listed concentrations were final and calculated for a 20 µL sample. All ASRE components were prepared in water, except PA, which was dissolved in 100 mmol L−1 sodium phosphate. The resulting samples were incubated for 30 min at 37 °C. After incubation, the reaction mixtures were subjected to 0.6% agarose gel electrophoresis. Integrated densities of all pBR322 forms in each lane were quantified using Image Studio analysis software (LI-COR Biotechnology, Bad Homburg, Germany) to estimate pDNA cleavage efficiency.
8
Fluorescein-Labeled DNA Damage Analysis
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A fluorescein (FAM)-labeled DNA duplex (Nick) was obtained by hybridization of 5′-FAM- CCGCTATTTCAACCCTTTGCAGTCCCAGAAGG-3′ with complementary oligonucleotides (5′-GGCGATAAAGTTGGG-3′ and 5′-pAAACGTCAGGGTCTTCC-3′) in a 1.0:1.5:1.5 ratio. The oligonucleotide mixture was incubated for 5 min at 95°C and then slowly cooled to room temperature.
A damaged pBR322 plasmid (New England BioLabs, Catalog #N3033L) was prepared by heat and acid treatment in combination with apurinic/apyrimidinic endonuclease 1 (APE1)-catalyzed cleavage of apurinic/apyrimidinic sites (Sukhanova et al., 2004 (link)).
A damaged pBR322 plasmid (New England BioLabs, Catalog #N3033L) was prepared by heat and acid treatment in combination with apurinic/apyrimidinic endonuclease 1 (APE1)-catalyzed cleavage of apurinic/apyrimidinic sites (Sukhanova et al., 2004 (link)).
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