RNase-Free DNase Set

RNeasy 96 Kit procedure requires two RNase-Fee DNase sets (cat. no. 79254) for each 96-well plate.

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.

To ensure efficient gDNA removal when doing an on-column digest using the RNase-Free DNase Set in combination with RNeasy Mini the following factors are crucial:

• prevent overloading by adjusting the amount of starting material to no more than the maximum amounts recommended in the RNeasy Mini Handbook
• ensure complete disruption and homogenization of the starting material as instructed in the section 'Disruption and homogenization of starting materials' of the handbook
• strictly follow the protocol for on-column DNase Digestion in Appendix D of the RNeasy Mini Handbook (you can let wash buffer RW1 incubate on the column for 3-5 minutes before centrifuging to enhance removal of excess gDNA prior to applying the enzyme)

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.