DNA extraction - Salting Out: Difference between revisions

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


Revision as of 21:05, 24 June 2008

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 isolate DNA from diverse 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

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 DNA
  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 70% ethanol, dry and resuspend in water or TE

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

You may wish to kill the Proteinase K (95°C, 10 minutes)

Notes

  • I routinely rely on the denaturation step of Hot-start Taq in my PCR to take care killing proteinase K (doesn't seem to effect TAq in this case)
  • Successfully used on fish, mammal and insect tissue.
  • Works well in 96 well plate format, to remove ethanol following precipitation/wash simply invert the plate over a sink then spin gently (~100 rcf) inverted over absorbant paper.

Acknowledgments

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

References

  • Miller SA, Dykes DD, Polesky HF (1988) A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Research, 16, 1215–1215.
  • Murray MG and Thompson WF (1980) Rapid isolation of high molecular weight plant DNA. Nucleic Acids Research, 8(19), 4321-4326.

See Also

Discussion

You can discuss this protocol.

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