New England Biolabs, M0301L, Topoisomerase I (E. coli)

Related Categories DNA Repair Enzymes and Structure-specific Endonucleases Specification Unit Definition One unit is defined as the amount of enzyme that catalyzes the relaxation of > 95% of 0.5 μg of pUC19 RF I (negatively supercoiled) DNA in 15 minutes at 37°C in a total reaction volume of 25 μl. DNA supercoiling is assessed by agarose gel electrophoresis in the absence of ethidium bromide. Reaction Conditions 1X rCutSmart™ Buffer Incubate at 37°C 1X rCutSmart™ Buffer 50 mM Potassium Acetate 20 mM Tris-acetate 10 mM Magnesium Acetate 100 µg/ml Recombinant Albumin (pH 7.9 @ 25°C) Activity in NEBuffers Storage Buffer 10 mM Tris-HCl 35 mM (NH4)2SO4 50 mM KCl 1 mM DTT 0.1 mM EDTA 50% Glycerol pH 7.5 @ 25°C Heat Inactivation 65°C for 20 minutes FAQ Q: Does Topoisomerase I relax all supercoiled DNA to the same degree or are the products an equilibrium of various conformations? A: Topoisomerase I will relax negatively supercoiled plasmids. There will be some variation in the linking number of relaxed DNA molecules, which will be distributed around the most preferred conformation. The difference in energy between DNA molecules with different linking numbers is quite small and they will distribute around a mean. Therefore, on an agarose gel without ethidium bromide, a distribution of bands will be seen even for a complete reaction. These bands should all still run slowly and are easily distinguished from supercoiled plasmids. Q: If the DNA resulting from a Topoisomerase I reaction is run in an ethidium bromide gel and there seems to be no evidence of enzyme activity, what is the most probable explanation? A: Ethidium bromide intercalates between the bases of the DNA and induces supercoiling. All relaxed plasmids regardless of linking number will become supercoiled in the presence of ethidium bromide and appear to run at the same position equivalent to any supercoiled DNA in the sample. Therefore to check the result of a Topoisomerase I reaction, the products MUST be run in a gel without any ethidium bromide. After the gel is run, it can then be stained with ethidium bromide to visualize the DNA. The relaxed forms of the plasmid will run slower and at the top of the gel. Any supercoiled plasmid will run faster and lower on the gel. Q: What is the molecular weight of Topoisomerase I? A: The molecular weight of Topoisomerase I is 97.4 kDa. Q: Can Topoisomerase I relax both positively and negatively supercoiled DNA? A: No. E. Coli Topoisomerase I can only relax negatively supercoiled DNA. Q: Does E. coli Toposiomerase I have the same properties as the Vaccinia Topoisomerase I? A: No. Topoisomerase I from E.coli can only relax negatively supercoiled DNA. Topoisomerase I from Vaccinia can relax both negatively and positively supercoiled DNA. Q: Will Topoisomerase I produce single strand-breaks in a plasmid? A: Topoisomerase I relaxed plasmids will not contain single-strand breaks. Any pre-existing nicked plasmids will remain as nicked plasmids in the sample. Q: Can you tell me more about the switch from BSA to Recombinant Albumin (rAlbumin) in NEBuffers? A: NEB is excited to announce that we have switched BSA-containing reaction buffers (NEBuffer 1.1, 2.1, 3.1 and CutSmart® Buffer) to Recombinant Albumin-containing buffers (NEBuffer r1.1, r2.1, r3.1 and rCutSmart™ Buffer). We are also in the process of moving all restriction enzyme formulations to contain rAlbumin. We feel that moving away from animal-containing products is a step in the right direction and are able to offer this enhancement at the same price.