{"product_id":"neb-m0356s","title":"New England Biolabs, M0356S, RNA 5' Pyrophosphohydrolase (RppH)","description":"RNA 5´ Pyrophosphohydrolase (RppH) removes pyrophosphate from the 5´ end of triphosphorylated RNA to leave a 5´ monophosphate RNA.  \u003cb\u003eRelated Categories\u003c\/b\u003e RNA Modification  \u003cb\u003eApplications\u003c\/b\u003e RNA Cloning  \u003cb\u003eSpecification\u003c\/b\u003e \u003cb\u003eUnit Definition\u003c\/b\u003e One unit is the amount of enzyme that converts 1 µg 300 mer RNA transcript into a XRN-1 digestible RNA in 30 minutes at 37°C.  \u003cb\u003eReaction Conditions\u003c\/b\u003e 1X NEBuffer™ 2 Incubate at 37°C 1X NEBuffer™ 2 50 mM NaCl 10 mM Tris-HCl 10 mM MgCl2 1 mM DTT (pH 7.9 @ 25°C)  \u003cb\u003eStorage Buffer\u003c\/b\u003e 20 mM Tris-HCl 200 mM NaCl 1 mM DTT 0.1 mM EDTA 50% Glycerol 0.01% Triton® X-100 pH 7.5 @ 25°C  \u003cb\u003eFAQ\u003c\/b\u003e Q: Can RppH replace Tobacco Acid Pyrophosphatase (TAP) as a decapping enzyme? A: The primary activity of RppH is to remove pyrophosphates from the 5’ end of triphosphate RNA. However, RppH does have decapping activity as well and will leave a 5' monophosphate when incubated with capped RNA. Here is a link to the protocol for this application. https:\/\/www.neb.com\/protocols\/2015\/01\/14\/decapping-eukaryotic-mrna-with-rpph-neb-m0356. There is a reference at the bottom of the protocol that highlights the decapping activity of RppH. Although we have not done a direct comparison between RppH and TAP in-house we have received significant positive feedback from customers using the enzyme in place of TAP. Note that customers using the decapping protocol will need to purchase the 10X Thermopol Buffer (NEB#B9004) separately. The enzyme is sold with NEBuffer 2 which is ideal for its pyrophosphate removal activity. Reference: Hetzel J, Duttke SH, Benner C, Chory J. Nascent RNA sequencing reveals distinct features in plant transcription. Proceedings of the National Academy of Sciences. 2016;113:12316–21. Neri F, Rapelli S, Krepelova A, Incarnato D, Parlato C, Basile G, et al. Intragenic DNA methylation prevents spurious transcription initiation. Nature. 2017;543:72–7. Q: Can RppH be heat inactivated? A: Yes, RppH can be heat inactivated by incubating at 65?°C for 20 minutes. However, heat treatment is not recommended due to the risk of RNA degradation. Alternative methods, such as RNA clean up or EDTA quenching, are preferred for preserving RNA integrity. Q: Is RNA 5' Pyrophosphohydrolase (RppH) salt tolerant? A: We recommend maintaining the reaction conditions below 200 mM salt. Q: Is it possible to digest RNase R (NEB #M0100)-resistant linear RNAs? A: Yes, we have tested two methods to digest RNase R-resistant linear RNAs: We recommend adding 1 unit of RNase R, 1 unit of XRN-1 (NEB #M0338), and 5 units of RppH (NEB #M0356) to 1 µg of RNA sample in 1X RNase R Reaction Buffer. The reaction should be incubated for 15 or 30 minutes at 37°C for efficient linear RNA removal. Pre-treat RNA samples with E. coli Poly(A) Polymerase (NEB #M0276) to lengthen the 3´ ends of the RNAs, which can improve RNase R binding and activity. The RNA needs to be cleaned up before proceeding with the RNase R reaction. ","brand":"New England Biolabs","offers":[{"title":"Default Title","offer_id":46835532857513,"sku":"M0356S","price":0.99,"currency_code":"USD","in_stock":true}],"url":"https:\/\/iright.com\/products\/neb-m0356s","provider":"Iright","version":"1.0","type":"link"}