DNase Protocol

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==Overview==
==Overview==
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Protocol for the Ambion DNA-free DNase kit. This uses a DNase inactivating reagent and thus avoids the DNase heat inactivation step that makes me nervous.
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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.
==Materials==
==Materials==
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List reagents, supplies and equipment necessary to perform the protocol here. For those materials which have their own OWW pages, link to that page. Alternatively, links to the suppliers' page on that material are also appropriate.
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*The DNAfree kit contains the stuff you need: [https://products.appliedbiosystems.com/ab/en/US/adirect/ab;jsessionid=2LdHJLWLKLylLhcGty6LnMnXzP2qcg66GJJ0XTL7s2zKXPFyqwBN!1659201712?cmd=catNavigate2&catID=603181 Applied Biosystems]
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*THAWED 10X DNase Buffer
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*supply 1 (i.e. tubes of a certain size? spreaders?)
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*reagent 1
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*X μL reagent 2
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**component A (reagent 2 is made up of multiple components)
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**component B
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*equipment 1
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*equipment 2
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==Procedure==
==Procedure==
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#1) In order to DNase 20ug of RNA in a 100ul reaction, mixup the following: <br>
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#In order to DNase 20ug of RNA in a 100ul reaction, mix the following:
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##10X DNase buffer#10ul<br>
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::*10&mu;L 10X DNase buffer
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##dH20#78ul<br>
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::*78&mu;L dH<sub>2</sub>0
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##DNase#2ul<br>
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::*2&mu;L DNase
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##RNA(1ug/ul)#10ul<br>
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::*10&mu;L RNA(1ug/&mu;L)
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#Incubate at 37&deg; C for 20-30 min.
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#Step 2
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#Add 0.1 volume resuspended DNase Inactivation Reagent.
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#*Step 2 has some additional information that goes with it.  i.e. Keep at 4&deg;C.
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#Incubate 2 min at RT mixing every 30s.
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#Step 3
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#Centrifuge at 10,000xg for 1.5 min.
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##Step 3 has multiple sub-steps within it.
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#Transfer solution to a new tube avoiding pelleted Inactivation Reagent.
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##Enumerate each of those.
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==Notes==
==Notes==
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#List troubleshooting tips here.   
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*[[User:Smoore|Smoore]] 09:59, 3 December 2008 (EST)DNase I requires calcium as well as magnesiumIf 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.
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#You can also link to FAQs/tips provided by other sources such as the manufacturer or other websites.
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#Anecdotal observations that might be of use to others can also be posted here.   
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*[[User:Smoore|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 heatingHot + metal = degraded RNA.
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Please sign your name to your note by adding <font face="courier"><nowiki>'''*~~~~''':</nowiki></font> to the beginning of your tip.
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*Please sign your name to your note by adding <font face="courier"><nowiki>'''*~~~~''':</nowiki></font> to the beginning of your tip.
==References==
==References==

Current revision

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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

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.
  • 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.


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