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).
Protocol
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)
Additional Information
Guidance on Choosing Sample Input Amounts when using the Monarch Total RNA Miniprep Kit
General Guidelines for Successful RNA Purification using the Monarch Total RNA Miniprep Kit
Product Manual
