Common aliquot sizes for TC
1,000X Neurotrophic factor stocks
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 |
|---|---|
BDNF |
R&D Systems 248-BDB-050 |
CNTF |
R&D Systems 257-NT-050 |
GDNF |
R&D Systems 212-GD-050 |
FGF |
PeproTech 100-18B |
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 10 µL of 10kx master stock aliquots for long term -80°C storage
You’ll end up with ~50 of 10kx aliquots
For BDNF, CNTF, GDNF only
Make 90 µL of 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 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
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.
Fetal Bovine Serum (FBS) Aliquots
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
100X Pen-strep aliquots
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.