{"product_id":"neb-m0766s","title":"New England Biolabs, M0766S, Thymine-DNA Glycosylase (TDG)","description":"\u003cb\u003eRelated Categories\u003c\/b\u003e DNA Repair Enzymes and Structure-specific Endonucleases  \u003cb\u003eSpecification\u003c\/b\u003e \u003cb\u003eUnit Definition\u003c\/b\u003e One unit is defined as the amount of enzyme required to create an AP site from 1 pmol of a 60-mer oligonucleotide duplex containing a single T:G mispair within a CpG context* in a total reaction of 20 μl in 1 hour at 37°C. *A T:G mispair is prepared by having the T located at the 24th position of a 5′ FAM-labelled 60 mer ssDNA and a G at the 25th position of an unlabeled complement, annealed to form a duplex substrate.  \u003cb\u003eReaction Conditions\u003c\/b\u003e 1X TDG Reaction Buffer Incubate at 37°C 1X TDG Reaction Buffer 50 mM Potassium Acetate 20 mM Tris-acetate 10 mM Magnesium Acetate 0.25 mM TCEP 10 µg\/ml Recombinant Albumin (pH 7.9 @ 25°C)  \u003cb\u003eStorage Buffer\u003c\/b\u003e 10 mM Tris-HCl 500 mM NaCl 0.1 mM EDTA 1 mM DTT 50% Glycerol pH 7.4 @ 25°C  \u003cb\u003eHeat Inactivation\u003c\/b\u003e 65°C for 10 minutes  \u003cb\u003eFAQ\u003c\/b\u003e Q: Is Thymine-DNA Glycosylase (TDG) (NEB #M0766) a tagged protein? A: TDG has a C-terminal His tag. Q: What is the activity of Thymine-DNA Glycosylase (TDG) (NEB #M0766) in different NEBuffers? A: TDG has 50% activity in NEBuffer r1.1, 0% activity in NEBuffer r2.1, 0% NEBuffer r3.1, and 20% activity in rCutSmart™. However, when each of these buffers is supplemented with fresh 1 mM TCEP or DTT in the reaction, TDG activity is restored to 100%. Q: Is Thymine-DNA Glycosylase (TDG) (NEB #M0766) inhibited by salt? A: Thymine-DNA Glycosylase (TDG) performance becomes sub-optimal when salt concentrations reach 200 mM NaCl. Q: I do not want to use the provided Thymine-DNA Glycosylase (TDG) reaction buffer for my experiment. Do you have any suggestions on how I can optimize my buffer conditions? A: Thymine-DNA Glycosylase (NEB #M0766) works best in the presence of a reducing agent, so the addition of 1 mM fresh DTT or TCEP will greatly improve performance. In the presence of fresh reducing agent, TDG activity can tolerate up to 200 mM NaCl before activity suffers. Q: Can Thymine-DNA Glycosylase (TDG) (NEB #M0766) be heat-inactivated? A: Yes. TDG can be heat-inactivated at 65°C for 10 minutes. Q: What bases will Thymine-DNA Glycosylase (TDG) (NEB #M0766) recognize and remove? A: The main substrates of TDG are dU or dT mismatched with dG in duplex DNA, with a preference for a CpG context (1). In addition, TDG has been reported to have a wide substrate tolerance, including activity on 5-formylcytosine and 5-carboxylcytosine (oxidation products of 5mC)(2), 5-hydroxymethyluracil(3), 3, N4- ethenocytosine(4), 5-fluorouracil(5), and other modified derivatives(6). References: 1. Waters, T.R. and Swann, P.F. (1998). J Biol Chem. 273, 20007-14. 2. He Y.F. et al. Science. (2011). 333, 1303-07. 3. Baker D. et al. (2002) Chem Res Toxicol. 15, 33-9. 4. Saparbaev, M. and Laval, J. (1998). PNAS. 95, 8508-13. 5. Hardeland, U. et al. (2000). J Biol Chem. 275, 33449-33456. 6. Bennett M.T. et al. (2006). J Am Chem Soc. 128(38), 12510-9. Q: What substrate is used to test Thymine-DNA Glycosylase (TDG) (NEB #M0766)? A: The substrates used to test enzyme activity are listed under the unit definition for each enzyme. TDG is tested using a 60-mer dsDNA substrate containing a centrally located T:G mispair in a CpG context. ","brand":"New England Biolabs","offers":[{"title":"Default Title","offer_id":46835494977705,"sku":"M0766S","price":0.99,"currency_code":"USD","in_stock":true}],"url":"https:\/\/iright.com\/products\/neb-m0766s","provider":"Iright","version":"1.0","type":"link"}