New England Biolabs, M0526S, Sce PUS1

This is an Related Categories Epitranscriptome Analysis,, RNA Modification Specification Reaction Conditions 1X NEBuffer™ r1.1 Incubate at 30°C 1X NEBuffer™ r1.1 10 mM Bis-Tris-Propane-HCl 10 mM MgCl2 100 µg/ml Recombinant Albumin (pH 7 @ 25°C) Heat Inactivation 55°C FAQ Q: What is the effect of Mg2+ concentration on Sce PUS1 activity? A: For certain RNA templates we found that the absence of Mg2+ in the reaction buffer can have a stimulatory effect on Sce PUS1 activity, while it can have an inhibitory effect on others. On other RNA templates, Mg2+ may be required for efficient Sce PUS1 activity. Q: How can Sce PUS1 activity be improved when low concentrations of RNA are used? A: Addition of 10% PEG 300 may improve Sce PUS1 activity, especially when low RNA concentrations are used. Q: What effect does MnCl2 have on Sce PUS1 activity? A: MnCl2 at concentrations > 1 mM has an inhibitory effect on Sce PUS1. Q: What is the effect of NaCl concentration on Sce PUS1 activity? A: Sce PUS1 is sensitive to NaCl; concentrations > 50 mM will inhibit Sce PUS1 activity. Q: Can Sce PUS1 modify more than 1 µg of RNA in one reaction? A: Depending on the substrate, additional Sce PUS1 may be necessary to convert > 1 µg of RNA. However, we recommend setting up multiple reactions containing < 1 µg to ensure greater efficiency. Q: Can I scale up or scale down the reaction volume of the Sce PUS1 reaction? A: We observed a drop in Sce PUS1 activity in reaction volumes smaller or larger than 100 µl. Thus, we recommend a minimum reaction volume of 100 µl, and to using multiple 100 µl reactions if scaling up for additional substrate is needed. Q: Can I shorten or extend the incubation time of Sce PUS1 with target RNA? A: We found that the majority of Sce PUS1 targeted uridines are converted to pseudouridines after 2 hours of incubation. However, preferred target sites may be converted after shorter incubation times. Depending on the substrate, incubation for more than 2 hours can further increase the uridine to pseudouridine conversion ratio. 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.