DNA extraction - Salting Out

Curators

• Marcus McHale Email me through OpenWetWare

• 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

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

• 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

• Oneill Lab:DNA Extraction
• DNA Precipitation
• DNA extraction from tissue
• Mouse tissue lysis for genotyping
• Ethanol precipitation of nucleic acids
• Purification of DNA

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.

You can discuss this protocol.