Difference between revisions of "Miniprep kit Macherey-Nagel"
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* Grow cells over night in 5ml liquid LB medium | * Grow cells over night in 5ml liquid LB medium | ||
| − | * Spin down cells from 2 ml in 2ml tube at | + | * Spin down cells from 2 ml in 2ml tube at 14000×g for 1 min |
| − | * | + | * Remove supernatant. |
* Repeat with another 2ml in the same tube | * Repeat with another 2ml in the same tube | ||
| + | ** Keep the remaining 1ml of cells for freezing if required | ||
* Add 150µl resuspension buffer and re-suspend all cells | * Add 150µl resuspension buffer and re-suspend all cells | ||
| − | * Add 250µl lysis buffer (blue) and invert 5 | + | * Add 250µl lysis buffer (blue) and invert 5 times |
* Add 350µl neutralisation buffer and invert till the whole liquid is no longer blue | * Add 350µl neutralisation buffer and invert till the whole liquid is no longer blue | ||
| − | * Spin down the cell debris and genomic DNA at 20. | + | * Spin down the cell debris and genomic DNA at 20.000×g for 3 min |
| − | * Prepare the columns in tubes or on the [[Vacuum manifold]] | + | * 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 | + | * Spin down (1min at 13000×g) (or vacuum) and remove flow through |
| − | * Add 450µl washing solution and spin down or vacuum | + | * Add 450µl washing solution and spin down (or vacuum) |
* Remove flow through and spin down to remove last bits of liquid | * Remove flow through and spin down to remove last bits of liquid | ||
* Move column to 1.5ml collection tube. | * Move column to 1.5ml collection tube. | ||
* Add 30µl elution buffer AE | * Add 30µl elution buffer AE | ||
| − | * Incubate for | + | * Incubate for 2 min |
| − | * Spin down (1min at | + | * Spin down (1min at 13000×g) |
* Add 30µl elution buffer AE | * Add 30µl elution buffer AE | ||
| − | * Incubate for | + | * Incubate for 2 min |
| − | * Spin down (1min at | + | * Spin down (1min at 13000×g) |
* Label tube and trash column | * Label tube and trash column | ||
Revision as of 17:20, 3 January 2021
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 14000×g for 1 min
- Remove supernatant.
- Repeat with another 2ml in the same tube
- Keep the remaining 1ml of cells for freezing if required
- Add 150µl resuspension buffer and re-suspend all cells
- Add 250µl lysis buffer (blue) and invert 5 times
- Add 350µl neutralisation buffer and invert till the whole liquid is no longer blue
- Spin down the cell debris and genomic DNA at 20.000×g 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 13000×g) (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 2 min
- Spin down (1min at 13000×g)
- Add 30µl elution buffer AE
- Incubate for 2 min
- Spin down (1min at 13000×g)
- Label tube and trash column
