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Isolating Plasmid from Cells (Miniprep)
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* Pellet overnight culture by centrifuging 13,000g for 10 minutes. | * Pellet overnight culture by centrifuging 13,000g for 10 minutes. | ||
* Pipette out supernatent and discard. | * 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. | * Add 250μl resuspension buffer R3 and mix by pipetting up and down. | ||
* Transfer to capped tubes. | * Transfer to capped tubes. | ||
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* Add 350μl precipitation buffer. Invert until mixture is homogenous. | * Add 350μl precipitation buffer. Invert until mixture is homogenous. | ||
* Centrifuge 13,000g for 10 minutes. Place spin column into recovery tube. | * Centrifuge 13,000g for 10 minutes. Place spin column into recovery tube. | ||
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* Load supernatent into spin column and discard capped tube. | * Load supernatent into spin column and discard capped tube. | ||
* Centrifuge 13,000g for 1 minute. Discard supernatent. | * Centrifuge 13,000g for 1 minute. Discard supernatent. | ||
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)
- 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.
