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:
- 10ul 10X DNase buffer
- 78ul dH20
- 2ul DNase
- 10ul RNA(1ug/ul)
- 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.
- 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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Relevant papers and books
- Goldbeter A and Koshland DE Jr. . pmid:6947258.
- JACOB F and MONOD J. . pmid:13718526.
- Mark Ptashne. A genetic switch. Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press, 2004. isbn:0879697164.
- Who has experience with this protocol?
or instead, discuss this protocol.