Difference between revisions of "Miniprep/GET buffer"
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Revision as of 17:13, 15 May 2007
Cheap non-kit plasmid miniprep:
- take 1.8 ml from an overnight culture into a 2 ml centrifuge tube, spin down 1 minute at full speed, and discard the medium
- resuspend the bacterial pellet by vortexing with 100 ul of chilled (4C) GET solution
- lyse the bacteria by adding 200 ul of room temperature alkaline SDS, not longer than 5 minutes. Invert to mix: do not vortex. The solution should become clear.
- precipitate proteins, membranes, and chromosomal DNA with 150 ul chilled (4C) potassium acetate solution, mix by inverting gently.
- centrifuge at full speed 5 to 10 min
- transfer 400 ul of the clean supernatant into a new tube preloaded with 900 ul of 100% EtOH to precipitate the plasmid DNA
- place the tubes in the -80 freezer for 30 minutes
- centrifuge the precipitated plasmid DNA 10 to 15 minutes at full speed, and discard supernatant
- optional: wash the pellet with 70% EtOH, respin and discard the supernatant
- air dry the pellet for 10-15 minutes at room temperature with the open tube on the bench
- resuspend the plasmid DNA in 20 ul of TE. The DNA will contain RNA contamination, which can be removed by resuspending in TE with RNAse.
GET buffer: 50 mM glucose (MW 180), 10mM EDTA, 25 mM Tris-HCl pH 8
Alkaline SDS: 0.2 N NaOH, 1% SDS
Potassium acetate: 3 M potassium acetate, 1.8 M acetic acid, no pH adjustment