Consensus template

Curators

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Materials

Pipettes and tips 1.5ml microcentrifuge tubes

Reagents

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

Equipment

Incubator/Water Bath: preferably shaking Centrifuge: preferably refrigerated

Procedure

Add 5μL Proteinase K to each mL of Digestion Buffer Homogenise (or simply place) tissue in solution Incubate at 55°C for 1 hour to overnight

Centrifuge at maximum speed in a benchtop centrifuge for 2 minutes 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 Centrifuge (preferably at 4°C) at maximum speed in a benchtop centrifuge for 20 minutes Transfer supernatant to a new tube Add >2 volumes of 98% ethanol Invert to mix and incubate at -20°C for 30 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

Critical steps

After adding sodium acetate and centrifuging be careful not to transfer any of the white solid (SDS) into the fresh tube

Notes

This protocol has evolved with me from a protocol I recieved from Damien Broderick, a supervisor from Queensland Department of Primary Industries and Fisheries, and I am sure many others have a similar story, please share your optimisations.

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.

It might also be good to add an image to show the workflow and timescales for experiment planning.

Acknowledgments

Acnkowledge any help you had in development, testing, writing this protocol.

References

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Specific Protocols

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