Haynes Lab:Notebook/Engineering PC-TFs/2013/10/24: Difference between revisions
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==October 24, 2013== | ==October 24, 2013== | ||
<span style="font-size:70%"''Harvest cells'' | <span style="font-size:70%">''Harvest cells'' '''Pellet 1–5 ml of an overnight recombinant E. coli culture by centrifugation'''. The optimal volume of culture to use depends upon the plasmid and culture density. For best yields, follow the instructions in the note below. Transfer the appropriate volume of the recombinant E.coli culture to a microcentrifuge tube and pellet cells at 12,000 x g for 1 minute. Discard the supernatant. Note: For best results with recombinant E. coli grown in LB (Luria Broth), use 1–3 ml of culture for high copy plasmids or 1–5 ml of culture for low copy plasmids. | ||
# ''Resuspend'': cells Important Reminder: Verify that appropriate volume RNase A Solution was added to the Resuspension Solution. '''Completely resuspend the bacterial pellet with 200 µl of the Resuspension Solution. Vortex or pipette up and down to thoroughly resuspend the cells until homogeneous'''. Incomplete resuspension will result in poor recovery.Another rapid way to resuspend the cell pellets is to scrape the bottoms of the microcentrifuge tubes back and forth 5 times across the surface of a polypropylene microcentrifuge tube storage rack with 5 x16 holes.^3 | # ''Resuspend'': cells Important Reminder: Verify that appropriate volume RNase A Solution was added to the Resuspension Solution. '''Completely resuspend the bacterial pellet with 200 µl of the Resuspension Solution. Vortex or pipette up and down to thoroughly resuspend the cells until homogeneous'''. Incomplete resuspension will result in poor recovery.Another rapid way to resuspend the cell pellets is to scrape the bottoms of the microcentrifuge tubes back and forth 5 times across the surface of a polypropylene microcentrifuge tube storage rack with 5 x16 holes.^3 |
Revision as of 13:47, 25 October 2013
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October 24, 2013Harvest cells Pellet 1–5 ml of an overnight recombinant E. coli culture by centrifugation. The optimal volume of culture to use depends upon the plasmid and culture density. For best yields, follow the instructions in the note below. Transfer the appropriate volume of the recombinant E.coli culture to a microcentrifuge tube and pellet cells at 12,000 x g for 1 minute. Discard the supernatant. Note: For best results with recombinant E. coli grown in LB (Luria Broth), use 1–3 ml of culture for high copy plasmids or 1–5 ml of culture for low copy plasmids.
applications, use water or 5 mM Tris-HCl, pH 8.0, as an eluant. Centrifuge at 12,000 x g for 1 minute. The DNA is now present in the eluate and is ready for immediate use or storage at –20 °C. Note: If a more concentrated plasmid DNA preparation is required, the elution volume may be reduced to a minimum of 50 µl.
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