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Relaxed and Supercoiled Plasmid DNA Substrates

BHE16300237

This product includes 0.050 mg of relaxed plasmid DNA at 1 mg/ml in TE buffer

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+1 866.986.9598
Email
[email protected]
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Storage Buffer 10 mM Tris-HCl, 2 mM EDTA, pH 7.5
Protocol DNA supercoiling Assay Protocol
1. Reaction and sample preparation:
The total volume of each reaction mixture is 40 µl including: 24 µl of H2O, 4 µl of 10 x buffer, 4 µl of 10 x relaxed DNA (250 µg/ml relaxed plasmid DNA), 4 µl of 10 x enzyme, 4 µl of 10 mM ATP. Incubate the reaction mixture at room temperature for 60 min.
 
The assay reaction and detection require a free magnesium concentration between 2 mM to 10 mM. EDTA should be avoided. For S. aureus gyrase, a final concentration of 400 mM potassium glutamate should be added into the reaction buffer. A concentration of 50 to 100 nM S. aureus gyrase is used.
 
2. Assay
(1) Freshly prepare the H19 dye by dilution of 1 µl the 1500 x H19 dye stock with 1.5 ml of the 1 x H19 dilution buffer (1500 x dilution).
(2) Mix 250 µl of the freshly prepared H19 dye with each reaction solution (40 µl). Incubate the mixture at room temperature for 5 min.
(3) Measure the fluorescence intensity at 535 nm using the excitation wavelength at 485 nm.
 
Human Topoisomerase I DNA Relaxation Assay Protocol
1. Reaction and sample preparation: The total volume of each reaction mixture is 40 µl including: 28 µl of H2O, 4 µl of 10 x Buffer HT1, 4 µl of 10 x supercoiled DNA (250 µg/ml), 4 µl of 10 x human topoisomerase I (1000 U/ml). Incubate the reaction mixture at 37°C for 30 to 60 min.
 
Note: The final concentrations are 25 mM Tris-HCl, pH 7.4, 58 mM KCl, 0.25 mM DTT, 5 mM MgCl2, 0.25 mM EDTA, 15 µg/ml BSA, 25 µg/ml supercoiled plasmid DNA and 100 U/ml topoisomerase I. A negative control reaction can be the reaction mixture without the enzyme. If the assay temperature is lower than 37°C, a higher enzyme concentration will be needed.
 
2. Assay
(1) For each 10 assays, freshly dilute 2 µl of 1500 x H19 dye into 3 ml of 1x H19 dilution buffer. The 1x H19 dilution buffer is prepared by 10 fold dilution of the 10x H19 dilution buffer with water.
(2) Mix 250 µl of the freshly prepared 1 x H19 dye with each reaction solution (40 µl). Incubate the mixture at room temperature for 5 min.
(3) Measure the fluorescence intensity at 535 nm using the excitation wavelength at 485 nm.
Reference 1. Asha M.K. et al, In vitro anti-Helicobacter pylori activity of a flavonoid rich extract of Glycyrrhiza glabra and its probable mechanisms of action, Journal of Ethnopharmacology, Vol.145(2), pp.581-586 (2013).
2. Mora-Pale M et al, Antimicrobial mechanism of resveratrol-trans-dihydrodimer produced from peroxidasecatalyzed oxidation of resveratrol. Biotechnol Bioeng.Vol 112, pp2417–2428 (2015). 
Combination SKU Supplier No. Specifications Size Your price Quantity
BHE16300237-1 RDNA-050UG Relaxed plasmid DNA 0.050 mg $227.09
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BHE16300237-2 RDNA-1MG Relaxed plasmid DNA 1 mg $2872.79
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BHE16300237-3 SDNA-050UG Supercoiled plasmid DNA 0.050 mg $201.13
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BHE16300237-4 SDNA-1MG Supercoiled plasmid DNA 1 mg $2190.83
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