In-tube DNase I Treatment after Purification with the Monarch Total RNA Miniprep Kit (NEB #T2010)
Materials and Equipment
- Reagents supplied by user: RNase-free microfuge tubes
Before you Begin
- For the 50 prep kit, add 275 μl nuclease-free water to the lyophilized DNase I vial and resuspend by gentle inversion. We suggest making aliquots of DNase I, sized to your processing needs, and storing at -20°C to minimize freeze-thaw cycles (3 F/T cycles maximum).
Sensitive applications such as RT-qPCR require that RNA input samples contain little to no genomic DNA. To further reduce any residual genomic DNA, you can treat your eluted sample with DNAse I in a separate reaction and clean up the sample using a second RNA Purification Column from this kit or by using a separate cleanup kit.
- For each sample to be treated, prepare a DNase I reaction mix in an RNase-free tube (not included) according to the table below. Mix well by gentle pipetting.
MIXTURE COMPONENT VOLUME RNA Sample (≤ 10 µg) Volume Adjusted with Water or TE Buffer 40 µl Monarch DNase I (reconstituted) 5 µl Monarch DNase I Reaction Buffer 5 µl TOTAL VOLUME 50 µl
- Incubate at room temperature (20–30°C) for 15 minutes.
- Proceed to RNA Reaction Cleanup using the Monarch Total RNA Miniprep Kit (NEB #T2010)