Latest revision as of 15:59, 28 October 2008

Newcastle University

GOLD MEDAL WINNER 2008

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Home >> Wet Lab >> Protocols >> Isolating Plasmid from Cells (Miniprep)

One of the centrifuges in our lab.

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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+{{:Team:Newcastle University/Template:UnderTheProtocol|page-title=[[Isolating Plasmid from Cells (Miniprep)]]}}
 * Pellet overnight culture by centrifuging 13,000g for 10 minutes.
 * Pipette out supernatent and discard.
+[[Image:Centrifuge.jpg|thumb|right|300px|One of the centrifuges in our lab.]]
 * Add 250μl resuspension buffer R3 and mix by pipetting up and down.
 * Transfer to capped tubes.