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| ==Materials== | | ==Materials== |
| Pipettes and tips
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| 1.5ml microcentrifuge tubes
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| ===Reagents=== | | ===Reagents=== |
| Digestion Buffer (pH 8.0): 10mM NaCl, 10mM TRIS (pH 8.0), 10mM EDTA (pH 8.0), 0.5% SDS,
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| Proteinase K 20mg/mL
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| Sodium Acetate 3M (pH 5.2)
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| Ethanol 70% and 98% (chill prior to use)
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| ===Equipment=== | | ===Equipment=== |
| Incubator/Water Bath: preferably shaking
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| Centrifuge: preferably refrigerated
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| ==Procedure== | | ==Procedure== |
| Add 5μL Proteinase K to each mL of Digestion Buffer
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| Homogenise (or simply place) tissue in solution
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| Incubate at 55°C for 1 hour to overnight
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| Centrifuge at maximum speed in a benchtop centrifuge for 2 minutes
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| Transfer supernatant into a new tube
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| Add 1/10 volume of Sodium Acetate 3M (pH5.2)
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| Invert to mix and incubate at -20°C for ~30 minutes
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| Centrifuge (preferably at 4°C) at maximum speed in a benchtop centrifuge for 20 minutes
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| Transfer supernatant to a new tube
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| Add >2 volumes of 98% ethanol
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| Invert to mix and incubate at -20°C for 30 minutes
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| Centrifuge (preferably at 4°C) at maximum speed in a benchtop centrifuge for 20 minutes
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| Wash pellet with 70% ethanol, dry and resuspend in water or TE
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| | ==Critical steps== |
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| ==Critical steps==
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| After adding sodium acetate and centrifuging be careful not to transfer any of the white solid (SDS) into the fresh tube
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| ==Troubleshooting== | | ==Troubleshooting== |
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| ==Notes== | | ==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.
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| Miller SA, Dykes DD, Polesky HF (1988) A simple salting out
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| procedure for extracting DNA from human nucleated cells.
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| Nucleic Acids Research, 16, 1215–1215.
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| It might also be good to add an image to show the workflow and timescales for experiment planning.
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| ==Acknowledgments== | | ==Acknowledgments== |
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Abstract
Materials
Reagents
Equipment
Procedure
Critical steps
Troubleshooting
Notes
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