Cat. No. / ID: 79254
The QIAGEN RNase-Free DNase Set is guaranteed RNase-free, quality-controlled, and optimized for use with RNeasy procedures and with QIAamp RNA Blood Mini procedures. Generally, DNase digestion is not required for RNA purified with RNeasy Kits since the silica-gel–membrane, spin-column technology efficiently removes the majority of the DNA without DNase treatment. However, more complete DNA removal may be necessary for certain RNA applications that are sensitive to very small amounts of DNA. Buffer RDD, included in the set, is optimized for on-column DNase digestion of 15 minutes at 20–30°C. The buffer is also well-suited for efficient DNase digestion in solution. The RNase-Free DNase Set provides efficient on-column digestion of DNA during RNA purification from cells and tissues using RNeasy Kits and the QIAamp RNA Blood Mini Kit. The DNase is efficiently removed in subsequent wash steps. For DNase treatment when using the RNeasy 96 Kit, please contact QIAGEN Technical Services or your local distributor for a separate, optimized protocol. The QIAGEN RNase-Free DNase Set is delivered as a stable, lyophilized enzyme. The RNase-Free DNase Set provides 1500 Kunitz units.
One Kunitz unit is defined as the amount of DNase I that causes an increase in A260 of 0.001 per minute per milliliter at 25°C, pH 5.0, with highly polymerized DNA as the substrate (Kunitz, M.  Crystalline desoxyribonuclease. I. Isolation and general properties, spectrophotometric method for the measurement of desoxyribonuclease activity. II. Digestion of thymus nucleic acid (desoxyribonucleic acid): the kinetics of the reaction. J. Gen. Physiol. 33, 349 and 363).
Yes. Even though buffer RDD in the RNase-Free DNase Set is optimized for on-column DNase digestion, the buffer is also well-suited for efficient DNase digestion in solution. Please note that the reaction must be cleaned up after the off-column DNase digest to remove the enzyme and buffer RDD, which will interfere with subsequent RT reactions.
A protocol for in-solution DNase digestion using the RNase-Free DNase Set can be found in Appendix C of the RNeasy MinElute Cleanup Handbook. For subsequent RNA Cleanup, use either the RNeasy MinElute Cleanup Kit, or follow the instructions for RNA Cleanup in the RNeasy Mini Handbook.
1. To obtain a concentration of 1.8 Kunitz/µl, dissolve each vial of lyophilized DNase in 833 µl of RNase-free water and combine the reconstituted enzyme solution from both vials into one vial.
2. Take 1 ml of this DNase I solution and mix with 7 ml of Buffer RDD* to prepare a DNase I incubation mix immediately before starting the RNeasy 96 protocol. Vortex briefly and keep on ice until use.
3. Use 80 µl of the DNase I incubation mix (from step 2 above) for each well of the RNeasy 96 plate, to perform on-column DNase digestion, according to instructions in the RNeasy 96 Handbook.
*The RDD Buffer Set for RNeasy 96 is available upon request. Please contact QIAGEN Technical Service for information on ordering. Buffer RDD from QIAGEN is optimized for DNase I digestion on the RNeasy membrane.
Carry out all procedures in a "DNA-free" workspace (see FAQ 2654). Be sure to include any DNase treatment steps in the recommended RNA isolation procedure or treat RNA separately with RNase-free DNase followed by repurification using a spin-column based method. RNeasy Mini Kit can be easily combined with RNase-free DNase. Alternatively, kits like the RNeasy Plus Universal Tissue already include a DNA removal step. Be sure to double both the units of enzyme and the incubation time recommended by the RNase-free DNase manufacturer. To minimize DNA contamination in your RNA preparations, and avoid the need for supplemental DNase treatments, we recommend using the RT2 First Strand Kit, which includes a highly efficient genomic DNA elimination step before reverse transcription.
NOTE: Our Chemistries are not compatible with AMBION's Turbo DNA-Free Kits.
In the rare case that trace amounts of genomic DNA are still detectable in sensitive downstream applications such as e.g., realtime RT-PCR, an in-solution digest using the RNase-Free DNAase set can be performed. Instructions are presented in Appendix C of the RNeasy MinElute Cleanup Handbook.
Alternatively, a second on-column digest can be carried out in future preparations, immediately following the RW1 wash after the first incubation with DNase.