DNA extraction - Salting Out: Difference between revisions

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==Procedure==
==Procedure==
#Add 5μL Proteinase K to each mL of Digestion Buffer
Tissue Digestion
#Add 5μL Proteinase K to each mL of Digestion Buffer (final 0.5mg/mL)
#Homogenise (or simply place) tissue in solution
#Homogenise (or simply place) tissue in solution
#Incubate at 55°C for 1 hour to overnight
#Incubate at 55°C for 1 hour to overnight
#Mix by vortexing then centrifuge at maximum speed in a benchtop centrifuge for 2 minutes
#Mix by vortexing then centrifuge at maximum speed in a benchtop centrifuge for 2 minutes
#Transfer supernatant into a new tube  
#Transfer supernatant into a new tube
#Add 1/10 volume of Sodium Acetate 3M (pH5.2)
 
#Invert to mix and incubate at -20°C for ~30 minutes
Precipitation of Protein and Cell Debris
#Add 1/10 volume of Sodium Acetate 3M pH 5.2 (final 0.3M)
#Invert to mix and incubate at -20°C for ~15 minutes
#Centrifuge (preferably at 4°C) at maximum speed in a benchtop centrifuge for 20 minutes
#Centrifuge (preferably at 4°C) at maximum speed in a benchtop centrifuge for 20 minutes
#Transfer supernatant to a new tube
#Transfer supernatant to a new tube
#Add >2 volumes of 98% ethanol
 
#Invert to mix and incubate at -20°C for 30 minutes
Precipitation of DNA
#Add >2 volumes of 98% ethanol (final 60-80%)
#Invert to mix and incubate at -20°C for ~15 minutes
#Centrifuge (preferably at 4°C) at maximum speed in a benchtop centrifuge for 20 minutes
#Centrifuge (preferably at 4°C) at maximum speed in a benchtop centrifuge for 20 minutes
#Wash pellet with 70% ethanol, dry and resuspend in water or TE
#Wash pellet with 70% ethanol, dry and resuspend in water or TE


==Critical steps==
==Critical steps==

Revision as of 20:44, 24 June 2008

Curators

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

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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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