Team:Paris/Miniprep

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Adaptation of PureYield(TM) Plasmid Miniprep System (Promega)

Prepare Lysate

• Put 2ml of bacterial culture in a 2ml microcentrifuge tube.
• Centrifuge 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 immediatly 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 the PureYield(TM) minicolumn without disturbing the cell debris pellet (yellow-orange).
• Apply vacuum pulling the lysate through the column.

Wash

• Add 200µl of Endotoxin Removal Wash (ERB) to the minicolumn. Allow the vacuum to pull the solution through the column.
• Add 400µl of Column Wash Solution (CWC) to the minicolumn. Allow the vacuum to pull the solution through the column. Release the vacuum, and remove the PureYield(TM) Minicolumn.
• Let dry for 5 minutes. (Preheat Nuclease Free Water at 37°C).

Elute

• Place the column in a 2ml collection tube, and centrifuge at maximum speed in a microcentrifuge for 2 minutes.
• Transfer the minicolumn into a clean 1.5ml microcentrifuge tube, then add 50µl of hot nuclease-free water directly to the minicolumn matrix. Let stand for 1 minute at room temperature.
• Centrifuge for 1 minute to eluate the plasmid DNA. Cap the microcentrifuge tube, and store eluted plasmid DNA at -20°C.