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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.
- The DNAfree kit contains the stuff you need: Applied Biosystems
- THAWED 10X DNase Buffer
- 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.
- 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.
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Relevant papers and books
- Goldbeter A and Koshland DE Jr. An amplified sensitivity arising from covalent modification in biological systems. Proc Natl Acad Sci U S A. 1981 Nov;78(11):6840-4. DOI:10.1073/pnas.78.11.6840 |
- JACOB F and MONOD J. Genetic regulatory mechanisms in the synthesis of proteins. J Mol Biol. 1961 Jun;3:318-56. DOI:10.1016/s0022-2836(61)80072-7 |
- Who has experience with this protocol?
or instead, discuss this protocol.