Isolated

Isolating Plasmid from Cells (Miniprep)

 * Pellet overnight culture by centrifuging 13,000g for 10 minutes.
 * Pipette out supernatent and discard.
 * Add 250μl resuspension buffer R3 and mix by pipetting up and down.
 * Transfer to capped tubes.
 * Add 250μl lysis buffer L7 and mix by inverting tube 5 times.
 * Incubate for 5 minutes at room temperature.
 * Add 350μl precipitation buffer. Invert until mixture is homogenous.
 * Centrifuge 13,000g for 10 minutes. Place spin column into recovery tube.
 * Load supernatent into spin column and discard capped tube.
 * Centrifuge 13,000g for 1 minute. Discard supernatent.
 * Add 700μl wash buffer W9 (with ethanol).
 * Centrifuge 13,000g for 1 minute. Discard supernatent.
 * Centrifuge 13,000g for 1 minute to remove all liquid. Discard any remaining supernatent and recovery tube.
 * Place spin column in new recovery tube.
 * Add 100μl TE buffer or MilliQ H2O.
 * Incubate for 1 minute at room temperature.
 * Centrifuge 13,000g for 2 minutes. The supernatent in the recovery tube should contain isolated plasmid.
 * Discard spin column and store plasmid at -20°C.

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