DNA extraction - Salting Out: Difference between revisions
From OpenWetWare
Jump to navigationJump to search
(→Notes) |
|||
Line 70: | Line 70: | ||
* [[DNA extraction from tissue]] | * [[DNA extraction from tissue]] | ||
* [[Mouse tissue lysis for genotyping]] | * [[Mouse tissue lysis for genotyping]] | ||
* [[Ethanol precipitation of nucleic acids]] | |||
* [[Purification of DNA]] | |||
==Discussion== | ==Discussion== |
Revision as of 01:25, 5 August 2008
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 isolate DNA from diverse tissues
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
- Vortex
Procedure
- Tissue Digestion
- Add 5μL Proteinase K to each mL of Digestion Buffer (final 0.5mg/mL)
- Homogenise (or simply place) tissue in solution
- Incubate at 55°C for 1 hour to overnight
- Mix by vortexing then centrifuge at maximum speed in a benchtop centrifuge for 2 minutes
- Transfer supernatant into a new tube
- Precipitation of Protein and Cell Debris
- Add 1/10 volume of Sodium Acetate 3M pH 5.2 (final 0.3M)
- Invert to mix and incubate at -20°C for ~15 minutes
- Centrifuge (preferably at 4°C) at maximum speed in a benchtop centrifuge for 20 minutes
- Transfer supernatant to a new tube
- Precipitation of Nucleic Acids
- Add ~2 volumes of 98% ethanol (final 60-80%)
- Invert to mix and incubate at -20°C for ~15 minutes
- Centrifuge (preferably at 4°C) at maximum speed in a benchtop centrifuge for 20 minutes
- 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
Troubleshooting
You may wish to kill the Proteinase K (95°C, 10 minutes)
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]
- I routinely rely on the denaturation step of Hot-start Taq in my PCR to take care killing proteinase K
- 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.
Acknowledgments
- Damien Broderick from Queensland Department of Primary Industries and Fisheries (Australia) first taught me this method.
References
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 (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 remove it, please make a note here.
You can discuss this protocol.