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Team:Warwick/Notebook/cellmaintenance
From 2014.igem.org
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| + | Materials: | ||
| + | 25ml DMEM media, 10ml PBS, 2ml Trypsin, (5ml Pen/strep, 50ml FBS). | ||
| + | 1 tissue culture plate, 1 15ml falcon tube. | ||
| + | Prep: | ||
| + | If using fresh DMEM, defrost pen/strep and 50ml FBS in 37⁰C waterbath (1 hour before starting). Then add 50ml FBS and 5ml pen/strep to DMEM media – label with name and date. | ||
| + | Warm DMEM, PBS and Trypsin in 37⁰C waterbath at least 30min in advance of starting. | ||
| + | Protocol: | ||
| + | |||
| + | <ul> | ||
| + | 1. Check cell confluency with microscope | ||
| + | 2. Aspirate off old media (with vacuum pump) | ||
| + | 3. Wash cells with 10ml warm PBS and aspirate PBS off | ||
| + | 4. Add 2ml Trypsin, move to incubator for 3-5min until cells begin to come off plate | ||
| + | 5. Check cells have detached from plate (microscope) | ||
| + | 6. Add 5ml DMEM media, wash plate thoroughly to re-suspend all cells, move to 15ml tube | ||
| + | 7. Spin 4min at 1000rpm and room temp | ||
| + | 8. Aspirate off media (do not disturb pellet) | ||
| + | 9. Re-suspend cells in 1ml DMEM media, add a further 9ml DMEM. Mix thoroughly. | ||
| + | 10. Plate 1ml culture and add 9ml DMEM (for a 1 in 10 dilution). | ||
| + | 11. Swirl plate side to side and up and down (not in a circular motion) | ||
| + | 12. Grow 2 days at 37⁰C and 5% CO¬2 | ||
| + | |||
| + | </ul> | ||
| + | |||
| + | Return DMEM and PBS to fridge, and pen/strep and trypsin to freezer. | ||
Revision as of 10:16, 5 August 2014
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