Haynes Lab:Notebook/Engineering PC-TFs/2013/10/22: Difference between revisions
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==October 22, 2013== | ==October 22, 2013== | ||
'''Materials''' | |||
* 70% ethanol spray bottle | |||
* 1x Phosphate-buffered saline (PBS) | |||
* Trypsin-EDTA buffer/ media | |||
* [http://openwetware.org/wiki/Haynes:MamCultureMedia Complete cell culture medium] (e.g., 10% FBS, 1% pen-strep) | |||
* Sterile tissue culture-treated vent-capped T-75 flask, one per new culture | |||
* 100% confluent (dense) cell culture (check confluency under the light microscope) | |||
'''Procedure''' | |||
# '''Pre-warm all liquid reagents to 37°C in the bead bath'''. | # '''Pre-warm all liquid reagents to 37°C in the bead bath'''. | ||
# After reagents are warmed, spray bottles down with 70% ethanol and prepare the biosafety hood as for routine work. Do all work in the biosafety hood. | # After reagents are warmed, spray bottles down with 70% ethanol and prepare the biosafety hood as for routine work. Do all work in the biosafety hood. | ||
# Retrieve the cell culture from the incubator. Stand the flask up | # Retrieve the cell culture from the incubator. Stand the flask up. Open the cap and aspirate old culture media from the bottom of the flask. | ||
# Add '''5 mL 1x PBS''' to the flask. Lay the flask flat (see image above) and wash by gently tilting the flask back and forth. | # Add '''5 mL 1x PBS''' to the flask. Lay the flask flat (see image above) and wash by gently tilting the flask back and forth. | ||
# Stand the flask up. Open the cap and aspirate off the PBS. | # Stand the flask up. Open the cap and aspirate off the PBS. | ||
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# Place the new flask into the 37°C incubator. The cells should adhere to the growth surface in a couple of hours. | # Place the new flask into the 37°C incubator. The cells should adhere to the growth surface in a couple of hours. | ||
# Discard the left-over cells as liquid waste by aspirating them into the vacuum trap. Discard all plastics, etc. that have come into contact with growth medium and cells as biohazard waste. | # Discard the left-over cells as liquid waste by aspirating them into the vacuum trap. Discard all plastics, etc. that have come into contact with growth medium and cells as biohazard waste. | ||
#Seed cultures in 6-well plates at ~2.5x105 so that cells will be ~5.0x105 at the time of transfection. | |||
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Latest revision as of 23:30, 26 September 2017
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October 22, 2013Materials
Procedure
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