Team:Penn/Protocols
From 2012.igem.org
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Revision as of 05:52, 30 September 2012
Protein Purification
RSB250-A Recipe:
- 20mM Tris
- 250mM NaCl
- 30mM Imidazole
- 10% Glycerol (v/v)
- 20mM Tris
- 250mM NaCl
- 500mM Imidazole
- 10% Glycerol (v/v)
Induction
- Grow a small booster culture in LB Broth overnight (~2-10% of final culture volume) w/ appropriate antibiotics
- Innoculate full scale culture & monitor OD600 every 15-30 minutes until OD600 reaches 0.8
- At OD600=0.8, induce with appropriate concentration of inducer (e.g. 1mM final concentration of IPTG for lac promoter)
- Allow to grow overnight @ 25°C
Pelleting & Sonication
- Collect cultures by centrifugation for 30 minutes @4°C & 5000g
- If sample volume is too large, repeat centrifugation (discarding the supernatant after each centrifugation)
- Resuspend pellet in RSB250-A (minimum 5% of culture volume)
- Pellet must be completely dissolved (no cell clumps) for efficient lysis.
- For especially difficult to resuspend pellets, sucking up and ejecting the cell suspension through a large gauge (narrow) needle several times can break up the pellet effectively
- Sonicate 5 cycles
- 1 cycle consists of 15 of lysis (10W power) followed by 5 seconds of cool down
- Lysis must be done on ice or excessive protein degradation will occur
- Spin lysate down @4°C & 20000g for 30 minutes
- Transfer supernatant into 15 mL Falcon tubes
- Add Ni-Agarose beads (1mL of beads per 100mL of culture volume)
- Wash beads 2-3 times with equal volumes of RSB250-A
- Spin down cells @ ~3000 rpm in a microcentrifuge
- Rotate overnight in a 4°C cold room
- Pour lysate and beads into a chromatography column, allow to empty through gravity flow
- Retain flow through (FT) on ice for later analysis
- Wash with 10 mL RSB250-A two times
- RSB250-A must be cold
- Collect wash fractions for later gel on ice
- Elute with 1% of culture volume of RSB250-B
- Collect elution fraction on ice
- Eluted protein can be stored at 4°C or -80°C
Thawing Mammalian Cells
- Prepare plates with 9mL of appropriate supplemented cell culture media
- Take the cells out from the -80 freezer
- Shake cells in a water bath at 37 degrees C
- Do this quickly, DMSO is extremely toxic to cells
- Spray this with tons of ethanol, dry with kimwipe, and put this in TC hood
- Add 1mL of media to the thawed cells, transfer immediately into plate
- Take 1 mL of media from the plate and wash the tubes to get any remaining cells
- Incubate overnight in cell incubator
- Passage cells 1:2
General Cell Culture
- Turn TC hood on to start flow and spray with 70% ethanol 30 min before culturing (see sterile technique protocol).
- You want to split cells when you see there is 60-70% confluency (under the microscope, 60-70% of the plate is filled with cells)
- Clean hands with 70% EtOH and rub them.
- Warm media, PBS, trypsin in a water bath at 37 degrees C for 15 minutes. Be sure not to exceed 15 min.
- Warm trypsin to room temperature (check on it periodically to not leave it out too long, because trypsin can degrade once warm).
- Aliquot the volume of media, PBS, trypsin into small test tubes depending on how much you need. Put the rest in the refrigerators you got them from
- Spray entire cell culture hood surface with 70% EtOH and wipe the cell culture hood
- Wipe from back of hood, working your way up to the front
- Aspirate your old cell culture media from cell culture plate (tip it to one side). Add 5 mL PBS for a 100mm dish. You can scale this with surface area for a smaller well. This is to wash off any extra proteins that are on the plate, because these can deactivate trypsin.
- Aspirate off the PBS
- Repeat wash
- Add 1 mL 0.25% trypsin for 100mm dishes
- Immediately deactivate trypsin with media. Add 9mL for a P100 plate.
- Wash plate until cells are suspended & no cell clumps are visible
- Seed into new plates in appropriate passage
- Label the new plate w/ date, name, dilution, cell type, & passage number (abbreviated P#)
- Shake plate on surface left-right and then front-back to evenly spread out the cells
- Observe seeded cells and verify they are evenly seeded
- Put seeded cell plate back in incubator
- Throw old plate away in biohazard
- Remove all bottles, place everything but the PBS back into the 4C refrigerator
- Spray entire cell culture hood surface
- Wipe from back of hood, working your way up to the front
- Make sure you change media every 2-3 days because the cells use up the nutrients, glucose, etc.