DNA Precipitation

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{{back to protocols}}
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==Overview==
==Overview==
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Replace this sentence with a brief description of the protocol and its goal.
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This protocol can be used to concentrate DNA, or to change the buffer the DNA is suspended in.  It can also be coupled with phenol chloroform extraction for the purifying nucleic acids.  This protocol also works for RNA precipitation (take care to use RNAse free materials in this case).
==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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*supply 1 (i.e. tubes of a certain size? spreaders?)
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*3M NaOAc pH 5.2
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*reagent 1
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*EtOH 95%
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*X μL reagent 2
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*Glycogen (optional)
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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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#Step 1
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# Add 0.1 volumes of 3M Sodium Acetate solution to 1 volume of DNA sample.
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#Step 2
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# Add 1ul Glycogen to the DNA sample.
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#*Step 2 has some additional information that goes with it. i.e. Keep at 4°C.
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# Add 2 volumes of 95% EtOH to the DNA Sample.
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#Step 3
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# Store the solution overnight at -20°C or for 30 minutes at -80°C.
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##Step 3 has multiple sub-steps within it.
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# Centrifuge the solution at maximum speed for least 15 minutes.
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##Enumerate each of those.
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# Decant and discard the supernatant.
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# (Optional) Add 1 ml of 70% EtOH to the pellet and let sit for 5 minutes.
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# (Optional) Centrifuge the sample at maximum spped for 5 minutes.
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# (Optional) Decant and Discard the supernatant.
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# Air-dry the pellet for 10-15 minutes at room temperature until all liquid is gone.
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# Resuspend in desired volume of water or buffer
==Notes==
==Notes==
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#List troubleshooting tips here. 
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*'''[[User:Ryan R Hurtado|Ryan R Hurtado]] 17:21, 22 April 2008 (EDT)''':
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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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-20°C for an hour is fine for using larger (1mL of bacterial culture, plasmid) amounts of DNA
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#Anecdotal observations that might be of use to others can also be posted here. 
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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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The DNA pellet will not always be visible depending on how much DNA you are precipitating.  So always take care in loading your samples in the centrifuge to remember the direction they are facing.  The DNA pellet will be on the part of the tube facing the outside of the centrifuge.
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This protocol will precipitate all nucleic acids, not just DNA.  If you do not want RNA in your sample, one of the many ways to deal with it is to simply resuspend in TE + RNAse at the last step and leave it at room temperature for 15mins-1hr.
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==BioCoder version==
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Following is the DNA Precipitation protocol in BioCoder, a high-level programming language for expressing biology protocols. What you see here is the auto-generated text ouput of the protocol that was coded up in BioCoder (see Source code). More information about BioCoder can be found on my home page. Feel free to mail me your comments/ suggestions.[[User:Vaishnavi Ananth|Vaishnavi]]
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====Text Output====
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[[DNA Precipitation protocol]]
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====Source Code====
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[[DNA Precipitation protocol - source code]]
==References==
==References==
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'''Relevant papers and books'''
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==Links==
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<!-- If this protocol has papers or books associated with it, list those references here.  See the [[OpenWetWare:Biblio]] page for more information. -->
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*[http://bitesizebio.com/2007/12/04/the-basics-how-ethanol-precipitation-of-dna-and-rna-works/ basics and discussion]
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<biblio>
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#Goldbeter-PNAS-1981 pmid=6947258
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#Jacob-JMB-1961 pmid=13718526
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#Ptashne-Genetic-Switch isbn=0879697164
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</biblio>
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==Contact==
==Contact==
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*Who has experience with this protocol?
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[[Talk:{{PAGENAME}}|Discuss this protocol]].  
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or instead, [[Talk:{{PAGENAME}}|discuss this protocol]].  
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<!-- You can tag this protocol with various categories.  See the [[Categories]] page for more information. -->
<!-- You can tag this protocol with various categories.  See the [[Categories]] page for more information. -->
[[Category:Protocol]]
[[Category:Protocol]]
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[[Category:DNA]]
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[[Category:In vitro]]
[[Category:Needs attention]]  <!--Delete this line once the protocol is complete-->
[[Category:Needs attention]]  <!--Delete this line once the protocol is complete-->
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<!-- Move the relevant categories above this line to tag your protocol with the label
 
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[[Category:In vitro]]
 
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[[Category:In vivo]]
 
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[[Category:In silico]]
 
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[[Category:DNA]]
 
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[[Category:RNA]]
 
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[[Category:Protein]]
 
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[[Category:Chemical]]
 
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[[Category:Escherichia coli]]
 
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[[Category:Yeast]]
 
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-->
 

Current revision

back to protocols

Contents

Overview

This protocol can be used to concentrate DNA, or to change the buffer the DNA is suspended in. It can also be coupled with phenol chloroform extraction for the purifying nucleic acids. This protocol also works for RNA precipitation (take care to use RNAse free materials in this case).

Materials

  • 3M NaOAc pH 5.2
  • EtOH 95%
  • Glycogen (optional)

Procedure

  1. Add 0.1 volumes of 3M Sodium Acetate solution to 1 volume of DNA sample.
  2. Add 1ul Glycogen to the DNA sample.
  3. Add 2 volumes of 95% EtOH to the DNA Sample.
  4. Store the solution overnight at -20°C or for 30 minutes at -80°C.
  5. Centrifuge the solution at maximum speed for least 15 minutes.
  6. Decant and discard the supernatant.
  7. (Optional) Add 1 ml of 70% EtOH to the pellet and let sit for 5 minutes.
  8. (Optional) Centrifuge the sample at maximum spped for 5 minutes.
  9. (Optional) Decant and Discard the supernatant.
  10. Air-dry the pellet for 10-15 minutes at room temperature until all liquid is gone.
  11. Resuspend in desired volume of water or buffer

Notes

-20°C for an hour is fine for using larger (1mL of bacterial culture, plasmid) amounts of DNA

The DNA pellet will not always be visible depending on how much DNA you are precipitating. So always take care in loading your samples in the centrifuge to remember the direction they are facing. The DNA pellet will be on the part of the tube facing the outside of the centrifuge.

This protocol will precipitate all nucleic acids, not just DNA. If you do not want RNA in your sample, one of the many ways to deal with it is to simply resuspend in TE + RNAse at the last step and leave it at room temperature for 15mins-1hr.

BioCoder version

Following is the DNA Precipitation protocol in BioCoder, a high-level programming language for expressing biology protocols. What you see here is the auto-generated text ouput of the protocol that was coded up in BioCoder (see Source code). More information about BioCoder can be found on my home page. Feel free to mail me your comments/ suggestions.Vaishnavi

Text Output

DNA Precipitation protocol

Source Code

DNA Precipitation protocol - source code

References

Links

Contact

Discuss this protocol.

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