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Lab July 21 2009
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Revision as of 21:22, 17 October 2009 by Jasoncummer (Talk | contribs)
At 10AM the optical density (600nm) of the culture was .275
aiming for a .3 optical density, we left the culture shaking while we went to a meeting.
Returned at 11 to find we had just missed our target OD, it was at an OD of .335
Decided that was close enough, placed the 250 ml of culture into a flat bottomed centrifuge bottle and spun it at 5000rpm for 10 minutes at 4 degrees C.
Here is the detailed protocol we are following (from http://openwetware.org/wiki/TOP10_chemically_competent_cells):
* Inoculate 250 ml of SOB medium with 1 ml vial of seed stock and grow at 20°C to an OD600nm of 0.3
o This takes approximately 16 hours.
o Controlling the temperature makes this a more reproducible process, but is not essential.
o Room temperature will work. You can adjust this temperature somewhat to fit your schedule
o Aim for lower, not higher OD if you can't hit this mark
* Centrifuge at 3000g at 4°C for 10 minutes in a flat bottom centrifuge bottle.
o Flat bottom centrifuge tubes make the fragile cells much easier to resuspend
o It is often easier to resuspend pellets by mixing before adding large amounts of buffer
* Gently resuspend in 80 ml of ice cold CCMB80 buffer [buffer was a bit warmer than would have been ideal... let it sit on ice briefly but did not get it completely cold]
o sometimes this is less than completely gentle. It still works.
* Incubate on ice 20 minutes
* Centrifuge again at 4°C and resuspend in 10 ml of ice cold CCMB80 buffer.
* Test OD of a mixture of 200 μl SOC and 50 μl of the resuspended cells.
* Add chilled CCMB80 to yield a final OD of 1.0-1.5 in this test. [final OD of .63, so we must have lost cells on the way. not ideal.]
* Incubate on ice for 20 minutes
* Aliquot to chilled screw top 2 ml vials or 50 μl into chilled microtiter plates[into 500 ul aliquots + 3 100 ul aliquots for testing competence]
* Store at -80°C indefinitely.
o Flash freezing does not appear to be necessary
* Test competence (see below)
* Thawing and refreezing partially used cell aliquots dramatically reduces transformation efficiency by about 3x the first time, and about 6x total after several freeze/thaw cycles.
