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RNeasy Mini Handbook 10/2019
85
Appendix E: DNase Digestion of RNA before
RNA Cleanup
This protocol describes how to use the RNAse-Free DNase Set (cat. no. 79254) to digest
contaminating DNA in RNA solutions prior to RNA cleanup. DNase digestion can alternatively
be carried out during RNA cleanup (see Appendix D, page 82). For samples highly
contaminated with DNA, we recommend DNase digestion in solution, as it is more efficient
than on-column DNase digestion.
Important points before starting
Generally, DNase digestion is not required since RNeasy technology efficiently removes
most of the DNA without DNase treatment. However, further DNA removal may be
necessary for certain RNA applications that are sensitive to very small amounts of DNA
(e.g., TaqMan RT-PCR analysis with a low-abundant target).
Important: Do not vortex the reconstituted DNase I.
DNase I is especially sensitive to physical denaturation. Mixing should only be carried
out by gently inverting the tube.
Things to do before starting
Prepare DNase I stock solution before using the RNAse-Free DNase Set for the first time.
Dissolve the lyophilized DNase I (1500 Kunitz units) in 550 µl of the RNAse-free water
provided. To avoid loss of DNase I, do not open the vial. Inject RNAse-free water into
the vial using an RNAse-free needle and syringe. Mix gently by inverting the vial. Do not
vortex.
For long-term storage of DNase I, remove the stock solution from the glass vial, divide it
into single-use aliquots, and store at −30 to −15°C for up to 9 months. Thawed aliquots
can be stored at 2–8°C for up to 6 weeks. Do not refreeze the aliquots after thawing.
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