DNA extraction - Salting Out

Curators

Marcus McHale Email me through OpenWetWare

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 for DNA extraction using salt

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

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

Notes

1. This protocol has evolved with me and I am sure many others, please share your optimisations.
2. You may wish to kill the Proteinase K (95°C, 10 minutes) after the extraction though if you are proceding straight to PCR (and don't leave your reactions sitting around before thermal cycling) the denaturation step will take care of that

Acknowledgments

1. 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

You can discuss this protocol.

Tag this page with categories to allow easier indexing and searching. See Categories for information on existing categories.