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 using salt to extract and purify 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

Procedure

1. Add 5μL Proteinase K to each mL of Digestion Buffer
2. Homogenise (or simply place) tissue in solution
3. Incubate at 55°C for 1 hour to overnight
4. Centrifuge at maximum speed in a benchtop centrifuge for 2 minutes
5. Transfer supernatant into a new tube
6. Add 1/10 volume of Sodium Acetate 3M (pH5.2)
7. Invert to mix and incubate at -20°C for ~30 minutes
8. Centrifuge (preferably at 4°C) at maximum speed in a benchtop centrifuge for 20 minutes
9. Transfer supernatant to a new tube
10. Add >2 volumes of 98% ethanol
11. Invert to mix and incubate at -20°C for 30 minutes
12. Centrifuge (preferably at 4°C) at maximum speed in a benchtop centrifuge for 20 minutes
13. 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 (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 Depaqrtment of Primary Industries and Fisheries supplied me with his version of this protocol to start me off.

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.

Discussion

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