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:
Check cell confluency with microscope
Aspirate off old media (with vacuum pump
Wash cells with 10ml warm PBS and aspirate PBS off
Add 2ml Trypsin, move to incubator for 3-5min until cells begin to come off plate
Check cells have detached from plate (microscope)
Add 5ml DMEM media, wash plate thoroughly to re-suspend all cells, move to 15ml tube
Spin 4min at 1000rpm and room temp
Aspirate off media (do not disturb pellet)
Re-suspend cells in 1ml DMEM media, add a further 9ml DMEM. Mix thoroughly.
Plate 1ml culture and add 9ml DMEM (for a 1 in 10 dilution).
Swirl plate side to side and up and down (not in a circular motion)
Grow 2 days at 37⁰C and 5% CO2
Return DMEM and PBS to fridge, and pen/strep and trypsin to freezer.
"
