DNase Protocol

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Overview

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.

Materials

• The DNAfree kit contains the stuff you need: Applied Biosystems
• THAWED 10X DNase Buffer

Procedure

1. 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)

1. Incubate at 37° C for 20-30 min.
2. Add 0.1 volume resuspended DNase Inactivation Reagent.
3. Incubate 2 min at RT mixing every 30s.
4. Centrifuge at 10,000xg for 1.5 min.
5. Transfer solution to a new tube avoiding pelleted Inactivation Reagent.

Notes

• 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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References

Relevant papers and books

1. Goldbeter A and Koshland DE Jr. . pmid:6947258. PubMed HubMed [Goldbeter-PNAS-1981]
2. JACOB F and MONOD J. . pmid:13718526. PubMed HubMed [Jacob-JMB-1961]
3. Mark Ptashne. A genetic switch. Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press, 2004. isbn:0879697164. [Ptashne-Genetic-Switch]

Contact

• Who has experience with this protocol?

or instead, discuss this protocol.