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Team:Paris/Miniprep
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| - | ==Adaptation of | + | ==Adaptation of PureYield(TM) Plasmid Miniprep System (Promega)== |
| - | + | ===Prepare Lysate=== | |
| + | *Add 2ml of bacterial culture to a 2ml microcentrifuge tube. | ||
| + | *Centrifuge 1.5ml of bacterial culture for 30 seconds at maximum speed in a microcentrifuge. Discard the supernatant. | ||
| + | *Add 600µl of H<sub>2</sub>O (Gibco), and resuspend completely. | ||
| + | *Add 100µl of Cell Lysis Buffer (Blue), and mix by inverting the tube 6 times. | ||
| + | *Wait 2min but no more than 5min | ||
| + | *Add 350µl of cold (4-8°C) Neutralization Solution, and mix thoroughly by inverting. | ||
| + | *Centrifuge at maximum speed in a microcentrifuge for 10 minutes. | ||
| + | *Place a PureYield(TM) minicolumn on a Luer-Lok(R) adapter of a VacMan(R). | ||
| + | *Transfer the supernatant (~900µl) into a PureYield(TM) minicolumn without disturbing the cell debris pellet (yellow-orange). | ||
| + | *Apply vacuum pulling the lysate through the column. | ||
| + | <br> | ||
| + | ===Wash=== | ||
| + | plop | ||
Revision as of 16:05, 3 August 2009
Adaptation of PureYield(TM) Plasmid Miniprep System (Promega)
Prepare Lysate
- Add 2ml of bacterial culture to a 2ml microcentrifuge tube.
- Centrifuge 1.5ml of bacterial culture for 30 seconds at maximum speed in a microcentrifuge. Discard the supernatant.
- Add 600µl of H2O (Gibco), and resuspend completely.
- Add 100µl of Cell Lysis Buffer (Blue), and mix by inverting the tube 6 times.
- Wait 2min but no more than 5min
- Add 350µl of cold (4-8°C) Neutralization Solution, and mix thoroughly by inverting.
- Centrifuge at maximum speed in a microcentrifuge for 10 minutes.
- Place a PureYield(TM) minicolumn on a Luer-Lok(R) adapter of a VacMan(R).
- Transfer the supernatant (~900µl) into a PureYield(TM) minicolumn without disturbing the cell debris pellet (yellow-orange).
- Apply vacuum pulling the lysate through the column.
Wash
plop
