{"product_id":"neb-m0665s","title":"New England Biolabs, M0665S, Tth Argonaute (TtAgo)","description":"argonaute (TtAgo) is a programmable DNA-endonuclease which requires a short 5’-phosphorylated single-stranded DNA guide to target its activity to a specific corresponding sequence on a substrate. TtAgo introduces one break in the phosphodiester backbone of the complementary substrate sequence.  \u003cb\u003eRelated Categories\u003c\/b\u003e Argonautes  \u003cb\u003eSpecification\u003c\/b\u003e \u003cb\u003eDiluent Compatibility\u003c\/b\u003e Diluent B  \u003cb\u003eStorage Buffer\u003c\/b\u003e 10 mM Tris-HCl 300 mM NaCl 1 mM DTT 0.1 mM EDTA 50% Glycerol pH 7.4 @ 25°C  \u003cb\u003eHeat Inactivation\u003c\/b\u003e No  \u003cb\u003eFAQ\u003c\/b\u003e Q: Can I prepare functional guides for TtAgo from unmodified synthetic oligos? A: Yes. You can order unmodified oligonucleotides and phosphorylate the guides using NEB T4 Polynucleotide Kinase and the Protocol for creating 5′-Phosphorylated Guides from Unmodified Oligonucleotides for use with TtAgo. This method is suitable for high-throughput screens of guides ordered in 96-well PCR plate format. Q: Some guides do not seem to be working as well as others with TtAgo. What can be done? A: While we do observe that some guide sequences are more optimally utilized by TtAgo than others (please refer to GUIDElines for optimization of Tth Argonaute (TtAgo) reactions for detailed information and recommendations for designing successful guides), another potential source of problems can arise from incomplete 5’-phosphorylation of the guide. We have noticed that when this modification is ordered from synthetic oligonucleotide vendors, the level of phosphorylation and corresponding activity can vary greatly. To remedy this issue, we suggest one of two options: 1) order unmodified guides and phosphorylate the guides using NEB T4 Polynucleotide Kinase and the Protocol for creating 5′-Phosphorylated Guides from Unmodified Oligonucleotides for use with TtAgo or 2) if phosphorylated guides have already been ordered, simply carry out the same protocol in option 1 with NEB T4 Polynucleotide Kinase as a phosphorylation polishing step. In general, guides ordered with phosphorylation modifications perform well, however it should be noted that we observe guide oligos phosphorylated with NEB T4 Polynucleotide Kinase to perform as well or better than those ordered with a phosphorylation modification. Q: How can I reduce non-specific endonuclease activity with TtAgo? A: Non-specific activity can be observed with double-stranded substrates, as TtAgo exhibits a degradative activity in its apo-form (in the absence of a guide), known as “chopping” [Swarts, et al. 2017]. This argonaute activity is thought to be involved in the creation of new guides for cellular defense, but can be problematic in vitro. We have observed that some guide sequences, while functional in guiding TtAgo endonuclease activity, may not be efficiently loaded into the argonaute. Thus, the “chopping” activity can take preference over the guided activity. In these situations, we recommend pre-loading the guide with TtAgo before attempting a reaction. Mix the guide and argonaute in 1X NEB ThermoPol® Reaction Buffer and incubate at 75°C for 10 minutes. We recommend using guide in a 3–5 times molar excess over TtAgo to reduce non-specific activity. We have also observed that reducing the reaction temperature and (to a lesser extent) titrating the amount of magnesium in the reaction can impact the amount of non-specific activity observed. Q: Can I store TtAgo with a guide “loaded?” A: We do not recommend storing TtAgo with a guide “loaded.” While the “loaded” nucleoprotein complex should be stable for 1–2 days if stored properly at -20–4°C, the presence of ssDNA guide is detrimental to long-term stability and reliable guided-endonuclease activity. Mix TtAgo and ssDNA guide shortly before intended use and in proportion to the scale of the experiment you wish to perform. Store the complex on ice when not used immediately. Q: Is TtAgo active in other NEB buffers? A: TtAgo shows the highest level of activity in NEB ThermoPol® Reaction Buffer, however, significant activity is observed in several other standard NEB buffers as well. Suitable results may still be obtained in non-optimal buffers with extended reaction times. Q: Is TtAgo active at other temperatures? A: TtAgo is a thermophilic enzyme derived from Thermus thermophilus. While it is active across a range of temperatures (~65°C-85°C) (see figure below), we recommend carrying out routine reactions in the temperature range of 75–80°C. Q: How much Mg2+ [magnesium(II)] should be included in a typical reaction? A: NEB ThermoPol® Reaction Buffer contains 2mM Mg2SO4 (magnesium sulfate), which is suitable for most reactions carried out with TtAgo. Higher amounts of Mg2+ can improve TtAgo performance on some substrates, but may also lead to more aberrant, off-target activity. A similar trend of slightly higher overall performance accompanied by off-target activity is observed when using TtAgo with Mn2+ as with Mg2+, however Mn2+ has lower range (0.1 to 2mM) of working concentrations compared to Mg2+ (0.5 to 5mM). For more information, please refer to GUIDElines for optimization of Tth Argonaute (TtAgo) reactions. Q: What if I want to use TtAgo with Mn2+ [manganese(II)] instead of Mg2+ [magnesium(II)] as provided in ThermoPol® Reaction Buffer? A: TtAgo can utilize either Mg2+ or Mn2+ in its active site. The typical reaction conditions in the provided NEB ThermoPol® Reaction Buffer are amenable to spiking Mn2+ into the reaction in the presence of 2mM Mg2+ with similar effect as with Mn2+ alone. However, if no Mg2+ is desired in the reaction, a custom buffer can be prepared. We recommend using 20 mM Tris-HCl pH 8.8 buffer with low salt (approximately 10 mM NaCl or KCl) and adding Mn2+ to the desired concentration (typically in the range of 0.1–2mM). A similar trend of slightly higher overall performance accompanied by off-target activity is observed when using TtAgo with Mn2+ as with Mg2+, however Mn2+ has lower range (0.1 to 2mM) of working concentrations compared to Mg2+ (0.5 to 5mM). For more information, please refer to GUIDElines for optimization of Tth Argonaute (TtAgo) reactions. ","brand":"New England Biolabs","offers":[{"title":"Default Title","offer_id":46835519062185,"sku":"M0665S","price":0.99,"currency_code":"USD","in_stock":true}],"url":"https:\/\/iright.com\/products\/neb-m0665s","provider":"Iright","version":"1.0","type":"link"}