DMEM and Trypsin should be at RT before use. Add them to water bath prior to use
18 FEB 2021
Thawing HEK293 rrp6-3xflag cells (one cryo-tube) to one T75 flask ( ~50% conf at time of seeding)
Thaw in water bath ~1min
Spin cells at 1000g 5min
Remove supernatant
Add 1ml of DMEM and resuspend
Transfer cells to T75 flask
20 FEB 2021
Split cells from one T75 to five T175
Each T175 got 30ml DMEM
1ml trypsin-EDTA (0.25%) used and 5 ml DMEM
Every flask got ~1ml of cells
23 FEB 2021
Split cells from one T175 to four 500cm2 plates
For each 500cm2 plate add 70ml DMEM (already reduced amount-don't add lower)
Add 2ml trypsin
Incubate (37C) 3-4minutes
Add 10ml DMEM and resuspend (~10-15times)
Every plate got ~3ml of cells
24 FEB 2021
Exchange media with media+5ng/ml dox (~9.30am)
Made 8ml of 5mg/ml dox
To make 10ug/ml working concentration i added 4990ul DMEM & 10ul of 5mg/ml dox
Add 250ul (10ug/ml) dox to each bottle of DMEM (500ml)
Add ~50ml to each plate
25 FEB 2021
Cell harvesting (24h post Dox induction)
Before start, put cool PBS and falcon tubes on ice
Empty the medium to a large beaker
Add 10-20ml ice cold pbs.
Scrape the plates and collect pbs (contains the cells) to falcon tube
Add another 10ml and scrape, repeat as many times as needed until no cells remain
Before centrifuge: prechill centrifuge at 4C and make sure the volume in all the falcon tubes is constant
After centrifuge for 5min 1000g 4C, dispose pbs to large beaker
Add 4ml PBs to one flacon tube, empty the pellet from this falcon to the next one (dont disturb the pellet) and repeat this with all tubes
Once all pellets are in one tube, wash tubes' walls with 2ml pbs and transfer to the tube containing all the pellets
Centrifuge this tube (1000g,5min,4C)
Remove supernatant to large beaker
Add 10ml of pbs to the tube, slightly resuspend
Add the cells to the 20-30ml syringe (plug the end with a yellow plug) and place the syringe in a 50ml falcon tube without the plunger (cover with parafilm)
Centrifuge at 1000g 5min 4C
Carefully aspirate the pbs with the vacuum (in the cabinet), when coming close to cells be very careful not to suck cells (make sure there is not pbs)
Add some liqN to a smaller plastic beaker and uncup the syringe (careful not to drop cells to the walls of the beaker)
A grey 50ml falcon tube should be labelled and precooled in liqN
Once cell bbs are formed, using a funnel add them to the falcon tube (make sure the volume of liqN is less than the volume of the falcon tube)
Use the special tube cup with holes to remove liqN
Store at -80C freezer (don't close tube tightly for the first 24h)