Tissue culture jobs
- Updated by Jamie Yelland, 2023-10-09. This checklist covers the general chores and ordering required to maintain the TC lab.
Equipment checklist
Vacuum Traps
- If the liquid in the trap flask is over the fill line, add 100mL of 100% bleach, allow to sit for ≥1 hour. Solution should turn blue, then clear. Once the solution is clear, dump down the sink while flushing the sink with running water. Add 100mL of 100% bleach to the empty flask and replace it back in the vacuum line.
- Autoclave the waste collection flasks approximately every two months.
Biohazard waste
- Dispose of biohazard when it is ≥75% full. Check weekly.
- Waste can be brought down to the biohazard disposal room, on floor E. Bags should be labeled with room number "B3-135", "Subramaniam", and the date.
Incubators
- The incubators are dry; there is no need to replace nor treat water.
- Sterilize the dry bath pellets every 6-12 months by autoclaving.
Ethanol
- If empty, replace ethanol in the 5 gallon drum below the sink.
- To do this, bring the tank down to the glassware services center on floor D. They have a cupboard for ethanol tank refill. Leave the tank in there with a purchase form (available by the cupboard). Purchase form just needs to say "vendor part# = EtOH, quantity = 5 gallons, 95% ethanol (5-gallon can)" plus our room number and a PO#, and they'll figure it out. The filled can will be returned within a couple of days.
- Use project # 228808 (last updated 10-09-23)
Reagents checklist
If we have less than the minimum quantity for any of these, place an order for more. "NA" just means these aren’t very important, or that we have plenty, so not much need to monitor.
| Have/Need? |
Consumables |
Minimum quantity |
|
25 mL pipettes |
2 boxes |
|
10 mL pipettes |
2 boxes |
|
5 mL pipettes |
2 boxes |
|
2 mL pipettes |
2 boxes |
|
15 cm dishes |
5 sleeves |
|
10 cm dishes |
5 sleeves |
|
6 cm dishes |
NA |
|
3 cm dishes |
NA |
|
6-well plates |
1 box |
|
12-well plates |
NA |
|
48-well plates |
NA |
|
96-well plates |
NA |
|
T75 flasks |
NA |
|
T25 flasks |
NA |
|
Flow Tubes |
NA (take from main lab) |
|
Autoclaved cryo vials and caps |
NA (take from main lab) |
|
Filter tips |
NA (take from main lab) |
|
Sani-cloth sterile wipes |
1 package |
- Note: we can run out of 15 cm dishes very quickly if people are doing large library preps. Anyone planning to use a lot of plates at once should note this and order extra.
| Have/Need? |
Reagents |
Minimum quantity |
|
100% FBS |
3 frozen 50mL aliquots |
|
Puromycin (2mg/mL) |
5 x 1mL aliquots |
|
Blasticidin (10mg/mL) |
5 x 1mL aliquots |
|
Hygromycin (100mg/mL) |
5 x 1mL aliquots |
|
Doxicyline (20 mg/mL) |
5 x 50uL aliquots |
|
AP1903 |
5 x 50uL aliquots |
- Note: We should have 5+ 500mL bottles of FBS stored at -80C.
- When the -20C 50mL FBS aliqouts start running low, thaw one of these bottles overnight at 4C in the TC fridge (might take more than one night to thaw).
- Once thawed, aliquot ~47.5mL of this into 10-11 50mL conical tubes, to create new "50mL" aliquots. The reason we don't fill all the way to 50 mL is that the FBS expands when it refreezes, and can crack the tubes if there's not enough space.
- It's okay to have a bit less than 50mL added to each DMEM bottle (47.5mL into 500mL DMEM =~ 9% FBS, which is fine for our lines).
- See Make 2mg/mL Blasticidin protocol for general instructions for making human antibiotic stocks. (Note: we now make 10mg/mL blasticidin aliquots; just adjust the protocol volumes for your desired antibiotic concentration).