Highly purified E. coli DNA Gyrase enzyme is offered as a holoenzyme (contains both A,B subunits). This is an excellent reagent for supercoiling plasmids in vitro or for novel drug screens.
E. coli DNA Gyrase Quality Control Tests
Nuclease contamination: assayed by testing for linear KDNA and linear plasmid DNA formation. Incubation of 1 µg of catenated KDNA or supercoiled pUC19 DNA for 4 hrs. at 37°C (under gyrase assay conditions and with or without ATP).
Supercoiling assays are carried out using relaxed DNA under conditions specified in the protocol sheets provided with the product. We also perform assays using kDNA as substrate. For details on this decatenation/supercoiling assay please refer to our technical literature. One unit of gyrase activity will supercoil 0.2 ug of plasmid in 30 min at 37°C.
A 5X assay buffer and dilution buffer is included with the enzyme. These are also sold separately as 5X Gyrase Assay Buffer (TG4030) Gyrase Dilution Buffer (please inquire).
Purification of Gyrase A and B Subunits
DNA gyrase is shipped on dry ice and should be stored at -70°C. We also recommend that the enzyme be aliquoted after the first thaw (repeated rounds of freeze/thaw may cause loss of activity); the enzyme activity is stable for 1-3 days on ice.
Reviews and Citations
- Phillips JW, Goetz MA, Smith SK, Zink DL, Polishook J, Onishi R, Salowe S, Wiltsie J, Allocco J, Sigmund J, Dorso K, Lee S, Skwish S, de la Cruz M, Martin J, Vicente F, Genilloud O, Lu J, Painter RE, Young K, Overbye K, Donald RGK, Singh SB: Discovery of Kibdelomycin, A Potent New Class of Bacterial Type II Topoisomerase Inhibitor by Chemical-Genetic Profiling in Staphylococcus aureus. Cell Chemistry and Biology 2011, 18: 955-965. doi: 10.1016/j.chembiol.2011.06.011.