Size selective DNA precipitation
Curators
Kersten S. Rabe
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Abstract
A very fast and easy method for the size-selective removal of smaller DNA from larger fragments. By adjusting the PEG and MgCl2 concentration the range of precipitated DNA fragments can be adjusted.
Materials
Reagents
- DNA to be separated
- 30% (w/v) PEG 8000/30 mM MgCl2 (concentration of PEG 8000 can be varied to shift the size of the percipitated DNA)
- TE Buffer, pH 8.0 (10 mM TRIS-HCl, 1 mM EDTA, pH 8.0)
Equipment
- Centrifuge which can do up to 10.000 rcf (=g)
- Appropriate tubes for the centrifuge
- Pipettes
- Vortexer
Procedure
- Mix 50 μL of sample with 150 µL of TE
- Add 100 µL of PEG/MgCl2
- Vortex
- Centrifuge 15 min at 10.000 rcf at roomtemperature
- Carefully remove supernatant not to disturb the pellet, which will be invisible
- Dissolve the pellet in a appropriate amount of buffer of choice
Critical steps
- Before centrifugation mark the tubes in order to know where the pellet will be expected afterwards, as the pellet will be (nearly) invisible
Troubleshooting
Notes
This protocol is being mention in the manual for the Gateway recombinational cloning system based on published methods [1, 2, 3]
Acknowledgments
Acnkowledge any help you had in development, testing, writing this protocol.
References
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PMID=2030954
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PMID=6246357
Specific Protocols
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