DNase Protocol

From OpenWetWare

(Difference between revisions)
Jump to: navigation, search
(Notes)
(Materials)
Line 5: Line 5:
==Materials==
==Materials==
*THAWED 10X DNase Buffer
*THAWED 10X DNase Buffer
-
*
 
-
*X μL reagent 2
 
-
**component A (reagent 2 is made up of multiple components)
 
-
**component B
 
-
*equipment 1
 
-
*equipment 2
 
==Procedure==
==Procedure==

Revision as of 09:22, 3 February 2009

Contents

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

  • 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

  1. 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.


  1. You can also link to FAQs/tips provided by other sources such as the manufacturer or other websites.
  1. Anecdotal observations that might be of use to others can also be posted here.


Please sign your name to your note by adding '''*~~~~''': to the beginning of your tip.

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]
All Medline abstracts: PubMed HubMed

Contact

  • Who has experience with this protocol?

or instead, discuss this protocol.


Personal tools