Difference between revisions of "DNase Protocol"

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==Overview==
 
==Overview==
  

Latest revision as of 09:34, 23 February 2009

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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

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. An amplified sensitivity arising from covalent modification in biological systems. Proc Natl Acad Sci U S A. 1981 Nov;78(11):6840-4. PubMed ID:6947258 | HubMed [Goldbeter-PNAS-1981]
  2. JACOB F and MONOD J. Genetic regulatory mechanisms in the synthesis of proteins. J Mol Biol. 1961 Jun;3:318-56. PubMed ID:13718526 | 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]
All Medline abstracts: PubMed | HubMed

Contact

  • Who has experience with this protocol?

or instead, discuss this protocol.