Miniprep kit Macherey-Nagel
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Mini-prep of plasmids using kit (E. coli)
Using kit from Macherey-Nagel
- Grow cells over night in 5ml liquid LB medium
- Spin down cells from 2 ml in 2ml tube at 14000xg for 1 min
- remove supernatant.
- Repeat with another 2ml in the same tube
- Add 150µl resuspension buffer and re-suspend all cells
- Add 250µl lysis buffer (blue) and invert 5 time
- Add 350µl neutralisation buffer and invert till the whole liquid is no longer blue
- Spin down the cell debris and genomic DNA at 20.000xg for 3 min
- Prepare the columns in tubes or on the Vacuum manifold
- add liquid without any pellet (better to add less supernatant than accidentally add gDNA)
- Spin down (1min at 13000xg) or vacuum and remove flow through
- Add 450µl washing solution and spin down or vacuum
- Remove flow through and spin down to remove last bits of liquid
- Move column to 1.5ml collection tube.
- Add 30µl elution buffer AE
- Incubate for 2min
- Spin down (1min at 13000xg)
- Add 30µl elution buffer AE
- Incubate for 2min
- Spin down (1min at 13000xg)
- Label tube and trash column
