Protocol for DNase treatment of total RNA. This uses a DNase inactivating reagent and thus avoids the DNase heat inactivation step that makes me nervous.
- THAWED 10X DNase Buffer
- X μL reagent 2
- component A (reagent 2 is made up of multiple components)
- component B
- equipment 1
- equipment 2
- In order to DNase 20ug of RNA in a 100ul reaction, mix the following:
- 10μL 10X DNase buffer
- 78μL dH20
- 2μL DNase
- 10μL RNA(1ug/μL)
- Incubate at 37° C for 20-30 min.
- Add 0.1 volume resuspended DNase Inactivation Reagent.
- Incubate 2 min at RT mixing every 30s.
- Centrifuge at 10,000xg for 1.5 min.
- Transfer solution to a new tube avoiding pelleted Inactivation Reagent.
- List troubleshooting tips here.
- Smoore 09:59, 3 December 2008 (EST)DNase I requires calcium as well as magnesium. If you are adding DNase I to degrade DNA in an existing solution (like a lysate), having 0.5-1.0 mM CaCl2 seems to really help. I don't know if it is in the kit buffer.
- Smoore 09:59, 3 December 2008 (EST)If you are poor and rely on heating your samples to kill the DNase, make sure to chelate the metals with excess EDTA before heating. Hot + metal = degraded RNA.
- You can also link to FAQs/tips provided by other sources such as the manufacturer or other websites.
- Anecdotal observations that might be of use to others can also be posted here.
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