New England Biolabs, M7636S, DNA Gyrase (E. coli)

Related Categories Other,, DNA Modifying Enzymes & Cloning Technologies Specification Materials Required but not Supplied Nuclease-free Water (NEB #B1500) Unit Definition One unit is defined as the amount of enzyme required to convert 0.5 µg relaxed pUC19 plasmid to the supercoiled form in 1X Gyrase Reaction Buffer incubated for 15 minutes at 37°C in a total reaction volume of 30 μl. Reaction Conditions 1X Gyrase Reaction Buffer Incubate at 37°C Heat Inactivation 65°C for 20 minutes FAQ Q: Can DNA Gyrase (E. coli) (NEB #M7636) create supercoils in both closed and open circular DNA? A: DNA Gyrase (E. coli) can supercoil closed circular DNA, but not open circular DNA. Q: Can DNA Gyrase (E. coli) (NEB #M7636) act on linear DNA? A: No. DNA Gyrase (E. coli) specifically acts on closed circular DNA and does not introduce supercoils into linear DNA. Q: Is DNA Gyrase (E. coli) (NEB #M7636) compatible with transformation or transfection workflows? A: Yes. Supercoiled DNA generated by Gyrase is suitable for transformation and may enhance transfection efficiency in some systems. Q: Are there contaminants that inhibit DNA Gyrase (E. coli) (NEB #M7636) activity? A: Yes. Avoid EDTA, SDS, phenol, or other contaminants in DNA preparations, as they can inhibit enzyme function. Q: How should DNA treated with DNA Gyrase (E. coli) (NEB #M7636) be run on a gel? A: To check the result of a Topoisomerase or Gyrase reaction, the reaction should be run on a gel with gel running buffer that does NOT contain EtBr. The gel can be stained with EtBr afterwards. Q: Is the linking number (ΔLk) or the superhelical density (σ) that results from the DNA Gyrase (E.coli) (NEB #M7636) reaction fixed or random? A: The resulting linking number is neither random nor fixed. There is a distribution of molecules of differing numbers of twists, based around a mean number. This value can vary based on factors, such as pH, temperature, ionic strength, sequence, length, cruciform formation, etc. Q: Will DNA Gyrase (E. coli) (NEB #M7636) add additional negative supercoils in DNA that is already partially supercoiled? A: DNA Gyrase (E. coli) can add negative supercoils to a circular DNA, even if it is already partially supercoiled, until no further supercoils can be introduced. Q: Does E.coli DNA Gyrase work in other NEBuffers? A: E.coli DNA Gyrase does not perform well in other NEBuffers. It shows no activity in T4 DNA Ligase Reaction Buffer. When NEBuffers are supplemented with 1.75 mM ATP final concentration, the observed activity is: NEBuffer™ r1.1: 0% NEBuffer™ r2.1: 0% NEBuffer™ r3.1: 0% rCutSmart™ Buffer: 12.5%