RNase R (C007B-565991)

One unit converts 1μg of poly-r(A) into acid-soluble nucleotides in 10 minutes at 37℃ in 20mM Tris-HCl (pH8.0), 100mM KCl and 0.1mM MgCl2.Application：Circular RNA studies; linear RNA removal from circular RNA; alternative splicing studies; analysis and identification of intron lariat sequences, etc.Source：E.coli RNase R expressed and purified from E. coli.Enzyme storage buffer：50mM Tris-HCl (pH7.5 at 25℃), 200mM NaCl, 1mM DTT, 0.1mM EDTA, 50% (v/v) Glycerol, 0.1% (w/v) Triton X-100.10X RNase R Reaction Buffer: 200mM Tris-HCl (pH8.0 at 25℃), 1M KCl, 1mM MgCl2.Inactivation or inhibition：RNase R is heat-inactivated by incubation at 70℃ for 10 minutes.Precautions：RNase R activity is dependent on magnesium ion at a concentration of 0.1-1.0mM. Chelators such as EDTA will significantly reduce the activity of RNase R. If necessary, add MgCl2 additionally in the reaction mixture to a final concentration of 0.1mM at least.This product is for R&D only. Not for drug, household, or other uses.For your safety and health, please wear a lab coat and disposable gloves during the operation.Instructions for Use：1. Linear RNA digestion with RNase Ra. Set up the reaction on ice as follows:ReagentVolume/ConcentrationRNA5µg10X RNase R Reaction Buffer2µl5µlRNase R (20U/µl)1-3U/µg RNA1-3U/µg RNADEPC-treated Waterup to 20µlup to 50µlb. Mix the reaction mixture well and incubate at 37℃ for 10-30min.c. Incubate at 70℃ for 10min to terminate the reaction.Note 1: The amount of RNase R and the volume of the reaction mixture need to be optimized through experiments.Note 2: The RNase R digestion product after heat-inactivation can be used directly for RT-PCR and qPCR, etc.2. Purification and recovery of circular RNA by one of the following methods a. Phenol/chloroform extraction followed by ethanol precipitation.(a) Add Nuclease-free Water into the reaction mixture obtained at step 1c to a total volume of 180μl. Add 20μl of 3M sodium acetate (pH 5.2) or 20μl of 5M ammonium acetate, mix well. Add 200μl of phenol/chloroform mixture (1:1), vortex vigorously for 20-30s. Centrifugation at 12000rpm for 5-10min and aspirate the upper layer phase into a new centrifuge tube.(b) Precipitate RNA by adding double volume of absolute ethanol, place at -20℃ for at least 30 minutes, and centrifuge at 12000rpm at 4℃ for 5-10min.(c) Discard the supernatant and wash the pellet with 500μl of ice-cold 70% ethanol to fully remove the salt.(d) Dissolve RNA in DEPC-treated Water and store at -80℃.b. Use RNA purification column or magnetic beads for RNA purification and recovery. References:1. Suzuki, H. et al., Nucl Acids Res. 2006; 34(8):e63.2. Vincent, H.A. and Deutscher, M.P., J Biol Chem. 2006; 281(40):29769.

Specification: Free of DNase, RNA endonuclease and exonuclease.