Difference between revisions of "DNase Protocol"

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(Materials)
(Procedure)
Line 14: Line 14:
 
==Procedure==
 
==Procedure==
 
#In order to DNase 20ug of RNA in a 100ul reaction, mix the following:
 
#In order to DNase 20ug of RNA in a 100ul reaction, mix the following:
::*10ul 10X DNase buffer
+
::*10μL 10X DNase buffer
::*78ul dH<sub>2</sub>0
+
::*78&mu;L dH<sub>2</sub>0
::*2ul DNase
+
::*2&mu;L DNase
::*10ul RNA(1ug/ul)
+
::*10&mu;L RNA(1ug/&mu;L)
 
#Incubate at 37&deg; C for 20-30 min.
 
#Incubate at 37&deg; C for 20-30 min.
 
#Add 0.1 volume resuspended DNase Inactivation Reagent.
 
#Add 0.1 volume resuspended DNase Inactivation Reagent.

Revision as of 07:16, 24 May 2007

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
  • X μL reagent 2
    • component A (reagent 2 is made up of multiple components)
    • component B
  • equipment 1
  • equipment 2

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.
  2. You can also link to FAQs/tips provided by other sources such as the manufacturer or other websites.
  3. 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. 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. ISBN:0879697164 [Ptashne-Genetic-Switch]
All Medline abstracts: PubMed | HubMed

Contact

  • Who has experience with this protocol?

or instead, discuss this protocol.