DNA extraction - Salting Out

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Revision as of 01:50, 23 September 2009 by Vaishnavi Ananth (Talk | contribs)
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Contents

Curators

  • This protocol has evolved with me and I am sure many others, please share your optimisations.

Anyone should feel free to add themselves as a curator for this consensus protocol. You do not need to be a curator in order to contribute. The OpenWetWare community is currently discussing the idea of protocol curators. Please contribute.

Abstract

  • Simple procedure to purify nucleic acids from diverse animal tissues

Materials

  1. Pipettes and tips
  2. 1.5ml microcentrifuge tubes

Reagents

  1. Digestion Buffer (pH 8.0): 10mM NaCl, 10mM TRIS (pH 8.0), 10mM EDTA (pH 8.0), 0.5% SDS,
  2. Proteinase K 20mg/mL
  3. Sodium acetate 3M (pH 5.2)
  4. Ethanol 70% and 98% (chill prior to use)

Equipment

  1. Incubator/Water Bath: preferably shaking
  2. Centrifuge: preferably refrigerated
  3. Vortex (optional)

Procedure

  • Tissue Digestion
  1. Add 5μL Proteinase K to each mL of Digestion Buffer (final 0.5mg/mL)
  2. Homogenise (or simply place) tissue in solution
  3. Incubate at 55°C for 1 hour to overnight
  4. Mix by vortexing then centrifuge at maximum speed in a benchtop centrifuge for 2 minutes
  5. Transfer supernatant into a new tube
  • Precipitation of Protein and Cell Debris
  1. Add 1/10 volume of Sodium Acetate 3M pH 5.2 (final 0.3M)
  2. Invert to mix and incubate at -20°C for ~15 minutes
  3. Centrifuge (preferably at 4°C) at maximum speed in a benchtop centrifuge for 20 minutes
  4. Transfer supernatant to a new tube
  • Precipitation of Nucleic Acids
  1. Add ~2 volumes of 98% ethanol (final 60-80%)
  2. Invert to mix and incubate at -20°C for ~15 minutes
  3. Centrifuge (preferably at 4°C) at maximum speed in a benchtop centrifuge for 20 minutes
  4. Wash pellet with 98% ethanol, and once or twice with 70%. Allow to air dry then resuspend in water or 1xTE

Critical steps

  • After adding sodium acetate and centrifuging be careful not to transfer any of the white solid (cell debris and SDS) into the fresh tube
  • Ensure to dry the pelletted DNA completely before attempting to resuspend

Troubleshooting

Notes

  • Other Salts (NaCl, AmOAc, LiCl) may also be used. See here for an explanation of how ethanol precipitation works and a description of the roles of different salts [1][2]
  • I routinely rely on the denaturation step of Hot-start Taq in my PCR to take care killing proteinase K (unless it's just not carrying over through the precipitations, either way it hasn't been a problem for me in PCR downstream).
  • I have successfully used this protocol to prepare quality PCR template from fish, mammal and insect tissue.
  • Works well for high throughput (e.g. 96 well plate extractions). To remove ethanol following precipitation/wash steps simply invert the plate over a sink then spin gently (~100 rcf) inverted over absorbant paper towelling.
  • To increase the efficiency of the ethanol washes you may vortex to dislodge the pellet and re-centrifuge (5-10 minutes)

Acknowledgments

  • Damien Broderick from Queensland Department of Primary Industries and Fisheries (Australia) first taught me this method.

References

See Also

Discussion

  • I have noticed in some samples a top layer following addition of the salt that looks like protein or fat (white and fluffy) but does not precipitate with the salt. I always pipette from beneath this layer to remove the aqeous phase. If anyone knows more about this or has a way to make it precipitate with the cell debris, please make a note here.

BioStream version

Following is the DNA extraction - Salting Out protocol in BioStream, 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 BioStream (see Source code). More information about BioStream can be found on my home page. Feel free to mail me your comments/ suggestions.Vaishnavi

Text Output

DNA extraction - Salting Out protocol

Source Code

DNA extraction - Salting Out - source code

You can discuss this protocol.

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