FBS
We get our FBS from Hyclone. We purchase FBS that has been ES cell tested for germline transmission. You can purchase it either with or without heat inactivation. Our policy is to purchase it heat inactivated, (there is an additional charge per bottle) because the serum is then ready for immediate use.
If your lab heat chooses to inactivates the serum:
1. Thaw FBS and mix well
2. Heat inactivate at 56 C for 30 min (use another bottle of H2O with a thermometer in it. When the H2O reaches 56 C begin timing).
3. Aliquot FBS in 100 ml aliquots.
4. Store at -20 C
LIF
Aliquot into 200 ul /tube
Store at 4 C
Gelatin
.1 g gelatin in 100 ml H2O
autoclave (gelatin will not go into solution until autoclaved)
final concentration 0.1 % gelatin
Trypsin
We purchase trypsin at 2.5%. This bottle is thawed and aliquoted out into 1 ml aliquots. These are stored at -20 C.
We take a 1 ml aliquot and add to 40 mls of versene. We store this trypsin dilution for 1 week at 4 C and throw away at the end of 1 week.
Versene
1 Liter
10x PBS 100ml
EDTA-Na 0.2g
Phenol red 15 mg
pH to 7.2 bring up to 1 Liter with ddH2O
Aliquot 40 ml/bottle and autoclave for 50 min.
Store at 4 C
Glutamine
Thaw bottle and aliquot into smaller dilutions (i.e. 2-3mls) Store at -20 C
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