Difference between revisions of "ChIP/Formaldehyde crosslinking cells"
Latest revision as of 12:59, 22 August 2006
This is a method for formaldehyde fixing adherent mammalian cells for chromatin immunoprecipitation.
- For 500 mL:
- 450 mL of DMEM
- 50 mL of FBS
- 5 mL of Pen/Strep
- Need 50 mL of media per 175 cm2 flask, plus additional for washing cells.
- Thaw 500 mL bottle of FBS at 4°C, make 50 mL aliquots, store at –20°C.
- Thaw FBS aliquot in H2O bath prior to making media (~37°C.)
- Make media and filter sterilize into 500 mL plastic bottle.
- Prior to beginning work, spray down cabinet with 70% EtOH + wipe
- Spray + wipe trypsin bottles, etc before putting them into the cabinet
- Aspirate media from flasks
- (Optional) wash cells once with PBS
- Add 10 mL of Trypsin per flask. Swish to cover the bottom. Incubate at 37°C for 5 min.
- 5 min incubation is a good time to prep media.
- Add 10 – 20 mL of media to each flask. Wash the bottom by swishing/pipetting up and down. Collect media into 50 mL conical tubes. (Have 5 ready if doing 10 flasks.)
- Add an additional 10 mL of media to one flask. Again wash the side with the cell growth to collect additional cells. Transfer cells and media to the next flask. Use a new 10 mL aliquot after 5 flasks.
- Centrifuge to pellet cells: 10 min at ~2000 rpm. (On the ghetto analog speed dial, position arrow slightly past the 1/2 mark.)
- Prep (10) new flasks during spin. Put 45 mL of media in each.
- Aspirate media from 50 mL conicals. Resuspend all pellets in a total of 20 mL media. (Transfer between tubes as necessary.)
- Count cells: ~10 uL of culture… dye… counting slide… fill in the details.
- Transfer 2.0 mL of culture into a new 50 mL conical to be passaged forward.
- Add 50 mL of fresh media. Aliquot into prepared flasks, 5 mL of culture each.
- Swish cells in new media to cover the bottom of the flask.
- Place flasks in the incubator.
- Check incubator is set to 37°C and 5% CO2, H2O pan in bottom is full.
- To the remaining 18.0 mL of culture, add 180 uL of 30% formaldehyde solution.
- Incubate at room temperature for 10 min.
- Make 10 mL of 1M glycine stock during incubation.
- Add 2.25 mL of glycine to culture (final concentration = 0.125 M glycine) and vortex. Incubate 5 min at room temp.
- Spin at 4°C, 3K rpm, 3-5 min. Discard supernatant in the formaldehyde waste for mammalian culture.
- Resuspend cells in 10 mL of PBS. Transfer to 15 mL conical tube, labeled with date, cell type, cell count, and your initials.
- Spin down. Again decant supernatant into the formaldehyde waste.
- Freeze pellet at –80°C. (Beth’s –80° space = box 21. Far freezer on the left. Second shelf from top, second rack from the left. Duncan’s space is in the 1st freezer on the right, third shelf from the top, 2nd box from the left.)
- See alternative protocol here.