Full-length wild-type S. aureus gyrase subunits (GyrA and GyrB, used for enzymological studies), as well as the wild-type gyrase core fusion truncate (GyrB27-A56) and a fusion truncate containing a GyrAY123F mutation (used for structural studies) were expressed and purified as described previously.25 (link)Negatively supercoiled pBR322 DNA was prepared from Escherichia coli using a Plasmid Mega Kit (Qiagen) as described by the manufacturer. Positively supercoiled pBR322 DNA was prepared by treating negatively supercoiled molecules with recombinant Archaeoglobus fulgidus reverse gyrase.49 (link)–50 (link) The number of positive supercoils induced by this process is comparable to the number of negative supercoils in the original pBR322 preparations.49 (link) In the experiments that compared negatively and positively supercoiled DNA, the negatively supercoiled plasmid preparations were processed identically to the positively supercoiled molecules except that reaction mixtures did not contain reverse gyrase. Relaxed pBR322 plasmid DNA was generated by treating negatively supercoiled pBR322 with calf thymus topoisomerase I (Invitrogen) and purified as described previously.27 (link)Gepotidacin was provided by GlaxoSmithKline. Moxifloxacin was obtained from LKT Laboratories. Gepotidacin and Moxifloxacin were stored at 4 °C as 20 mM stock solutions in 100% dimethyl sulfoxide.
Calf thymus topoisomerase 1
Manufactured by Thermo Fisher Scientific
Calf thymus topoisomerase I is an enzyme that relaxes supercoiled DNA by introducing transient single-strand breaks. It can be used in various molecular biology applications that require the manipulation of DNA topology.
Automatically generated - may contain errors
Lab products found in correlation
Plasmid mega kit, by Qiagen (1 mentions)
Moxifloxacin, by LKT Laboratories (1 mentions)
Gepotidacin, by GlaxoSmithKline (1 mentions)
Phenol chloroform isoamyl alcohol, by Thermo Fisher Scientific (1 mentions)
G box, by Syngene (1 mentions)
Gelred fluorescent dye, by Biotium (1 mentions)
Wheat germ topoisomerase 1, by Promega (1 mentions)
Nb mva1269i, by Thermo Fisher Scientific (1 mentions)
4 protocols using calf thymus topoisomerase 1
1
Purification and Supercoiling of pBR322 DNA
2
Topological analysis of plasmid DNA binding to Hfq
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The experiments were performed as described in Tupper et al.45 (link). 5 µg of purified pHSG298 (2675 bp, KanR, Takara Bio) plasmid was incubated 10 min at room temperature with full length Hfq in a total volume of 100 µL of buffer containing 10 mM Tris-HCl pH 7.5, 0.1 mM EDTA, 15 mM KCl, 2 mM spermidine, 15% v/v glycerol and 0.1 g.L−1 BSA. 18U of calf thymus topoisomerase I (Invitrogen) were then added and the incubation continued for 30 min at 37 °C. Proteins were extracted with ultrapure phenol/chloroform/isoamyl alcohol (25:24:1 v/v, Invitrogen) and ethanol precipitation. The DNA was re-suspended in 50 µL Tris-HCl (5 mM, pH8). 2 µL of purified plasmids were electrophoresed in a 1% w/v native agarose gel. Electrophoresis was performed in Tris-Borate-EDTA 1X, 16.5 V/cm during 30 min. The gel was then rinsed 15 min with water before staining 30 min with GelRed fluorescent dye (Biotium, Inc.). Images were acquired with a G-box (Syngene) imager.
3
Topoisomerase I-Mediated DNA Supercoil Relaxation
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Ten microgram of supercoiled transcription templates were treated during 1 h at 37°C with 2 μl of calf thymus topoisomerase I (6 U/μl, Invitrogen) in buffer containing 50 mM Tris–HCl pH 7.5, 50 mM KCl, 10 mM MgCl2, 0.5 mM DTT, 0.1 mM EDTA, 30 μg/ml bovine serun albumin (BSA). After a phenol:chloroform:IAA (25:24:1) extraction, the DNA was precipitated with isopropanol and 0.3 M sodium acetate, washed with 70% ethanol, dried and resuspended in 25 μl RNase free-H2O. Topoisomerase I-treated plasmids were resolved, as described below, to observe the integrity of the DNA and the distribution of the different topoisomers.
4
Enzyme Selection for Optimal DNA Manipulation
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All enzymes were from New England Biolabs, with the exception of wheat germ topoisomerase I (Promega in early experiments, later Inspiralis), calf thymus topoisomerase I (Invitrogen), and nicking endonuclease Nb.Mva1269 I (Fermentas), which was preferred to its isoschizomer Nb.BsmI for its optimal digestion temperature of 37°C, as opposed to 65°C for Nb.BsmI.
Note: care was taken not to overdigest with Nb.BtsI, as this nicking enzyme tended to show non-specific star activity when used at high concentration (data not shown).
Note: care was taken not to overdigest with Nb.BtsI, as this nicking enzyme tended to show non-specific star activity when used at high concentration (data not shown).
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