Oct 16, 2019
DNase I treatment in solution (after RNA extraction)
- 1Wageningen University
- iGEM Wageningen 2019
Protocol Citation: Ben Kuipers 2019. DNase I treatment in solution (after RNA extraction). protocols.io https://dx.doi.org/10.17504/protocols.io.75ghq3w
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol in our group and it is working for at least gDNA. Plasmid DNA not.
Created: October 11, 2019
Last Modified: October 16, 2019
Protocol Integer ID: 28552
Keywords: RNA extraction
Abstract
This is a protocol used for the Dnase treatment after RNA isolation with the RNeasy mini kit. Protocol is based on the RNase-Free DNase Set and the RNeasy Mini Handbook 10/2013, Qiagen 'Appenix E: DNase Digestion of RNA before RNA Cleanup'.
Check the concentrations of DNA and RNA with Qubit.
DNase I Stock solution was made from the Promega Maxwell 16 LEV SimplyRNA kit DNase by adding 275 µL nuclease free water to the lyophilized DNase I.
Prepare a mastermix of 5 µL Buffer RDD (RNase-Free DNase Set) + 1.25 µL DNase I stock (Promega Maxwell 16 LEV simplyRNA kit) + eluted sample. Make the volume up to 50 µL with RNase-free water (RNase-Free DNase Set).
Incubate the mixture on room temperature for 60 minutes.
Check the concentrations of DNA and RNA with Qubit again.