Common aliquot sizes for TC¶
Neurotrophic factor stocks (10kx and 1kx)¶
Final concentration should be 10 ng/mL of each neurotrophic growth factor
Important
Dissolve neurotrophics into 0.1% BSA/PBS only, using BSA, Millipore Sigma, A2058. For the FGF, it is possible to do sterile PBS but it is recommended that you use the same filtered 0.1% BSA/PBS
Neurotrophic |
Cat |
Master stock aliquot |
Working stock aliquot |
|---|---|---|---|
BDNF |
R&D Systems 248-BDB-050 |
90 µL, 100 µg/ml (10kx) |
50 µL, 10 µg/ml (1kx) |
CNTF |
R&D Systems 257-NT-050 |
90 µL, 100 µg/ml (10kx) |
50 µL, 10 µg/ml (1kx) |
GDNF |
R&D Systems 212-GD-050 |
90 µL, 100 µg/ml (10kx) |
50 µL, 10 µg/ml (1kx) |
FGF |
PeproTech 100-18B |
45 µL, 1,000 µg/ml (100kx) |
25 µL, 100 µg/ml (10kx) |
For Neurotrophic factors, from the reagents in the glass tubes:
Make a 0.1% BSA/PBS (ONLY use Millipore Sigma, A2058) solution and syringe filter through a 0.2 µm filter
Add 500 µL filtered 0.1% BSA/PBS to a 50 µg bottle of neurotrophics to get a 100 µg/ml (10kx) master stock
For FGF only
Make 45 µL @100kx master stock aliquots for long term -80°C storage
For 1 mg, add 1 mL 0.1% BSA/PBS. You’ll end up with ~21 of 10kx aliquots
Directly add 405 µL filtered 0.1% BSA/PBS to the 45 µL 100kx master stock to make 100 µg/ml (10kx) stock aliquots
For the last 100kx master stock aliquot, you’ll have to manually measure it day of to know how much BSA/PBS to add. e.g. 35.8 µL will need 322.2 µL.
Make 25 µL @10kx aliquots for long term -80°C storage (don’t keep at -20°C!)
You’ll end up with ~18 of 10kx aliquots (2 rows of a cardboard box)
Don’t keep at -20°C because it lasts so long
For BDNF, CNTF, GDNF only
Make 90 µL @10kx master stock aliquots using for long term -80°C storage
You’ll end up with ~5.5 of 10kx aliquots
For BDNF, CNTF, GDNF only, directly add 810 µL filtered 0.1% BSA/PBS to the 90 µL 10kx master stock to make 10 µg/ml (1,000x) stock aliquots
For the last half 10kx master stock aliquot, you’ll have to manually measure it day of to know how much BSA/PBS to add. e.g. 35.8 µL will need 322.2 µL.
Make 50 µL @1kx aliquots for medium term -20°C storage
You’ll end up with ~18 of 1,000x aliquots (2 rows of a cardboard box)
Leave the last aliquot for short term use in 4°C
You can use the full 50 µL -20°C aliquots to directly make 50 mL N3 aliquots for reprogramming experiments
RepSox (1kx)¶
Property |
|
|---|---|
Selleck Chem # |
S7223 |
MW |
287.32 |
1000x stock |
7.5 mM |
Final concentration |
7.5 uM |
To make working 1,000x stock (7.5 mM), resuspend 25 mg RepSox in 11.6 mL DMSO.
Aliquot 50 uL in 600 uL tubes and store at -20°C in a labeled box.
Can make a 10kx stock also
Fetal Bovine Serum (FBS) Aliquots (50 mL)¶
FBS contains many nutrients and growth factors to support cell growth. To preserve FBS, it is usually stored in the -20°C freezer until used. To avoid multiple freeze-thaw cycles of FBS and to prevent contaminations we make FBS aliquots.
Store aliquots in -20°C freezer in the TC room.
Take responsibility for aliquoting new stocks, when older stocks are almost used up. Make sure to use sterile technique in preparing aliquots.
Thaw 500 mL FBS bottle in the fridge 2 days beforehand
Make ~10 aliquots of 50 mL each. Be sure to stop at the 50mL mark. Having larger volumes may cause the FBS to overflow (and be contaminated) when frozen again (FBS volume expands when frozen).
Make 12 mL aliquots with any leftover for 80% FBS/20% DMSO freezing media (just add 3 mL DMSO)
Note
This is now a lab job. As of 3/15/23, notify Nat if more aliquots need to be made.
Important
As of 3/15/23, we will ask Brad from Genesee to set aside an FBS lot and send us a sample size. Bulk ordering saves us $170/bottle (~$6k over year) and helps reduce lot-to-lot variability. Test old and new FBS in reprogramming. Use the sample FBS to defrost the MEFs, make the Plat-E virus, and do the infection until 4 dpi. Assess 14 dpi reprogramming between the two lots. You will have to heat inactivate the sample FBS as they do not heat inactivate small 50 mL FBS lot samples. You should freeze it at -20°C to match number of freeze-thaw cycles as the currrent FBS. An example of results
Pen-strep aliquots (100X, 5 mL)¶
Penicillin-streptomycin is a mixture of penicillin and streptomycin and is added to the media to prevent bacterial contamination. However, it is always important to be very careful when handling cultures as contamination may still occur even when pen-strep is added.
Thaw 50 mL pen-strep bottle in the fridge overnight
Pen-strep is aliquoted into 5 mL aliquots and stored in the TC -20°C until used.
Take responsibility for aliquoting new stocks, when older stocks are almost used up. Make sure to use sterile technique in preparing aliquots.
50X Geltrex aliquots¶
New orders of Geltrex (Thermo Fisher Scientific A1413302) should be stored at -80ºC until aliquoting
Thaw the new vial overnight at 4ºC
The next day, prepare ice (use designated TC styrofoam) to chill reagents during aliquoting
Pre-chill DMEM/F12 and fresh, labeled 1.7 mL microcentrifuge tubes on ice
Dilute the Geltrex 1:1 with the cold DMEM/F12. Immediately aliquot 120 µL each into prepared tubes.
To use, further dilute a 120 µL 50X Geltrex aliquot in 6 mL cold DMEM/F12 (for a final 1:100 dilution). This is enough to coat one 6-well plate. See Geltrex coating plates for additional details.