E0:
You will need:
- 500 ml bottle MEM
- 5 ml glutamine
- 5 ml MEM vitamin mix
- 10 ml non-essential amino acids
- 5 ml 10g/100ml glucose
- 5 ml PenStrep
- 0.5 ml Gentamycin
Add a few drops of the 1M NaOH to bring the pH back up to around 7.5-going from orange-red back to just short of blue-red.
Filter sterilize.
E5:
Add 5% volume of heat inactivated FBS (so for 250 mls, add 12.5 ml of FBS--I know that makes the final about 4.5% FBS not 5% but that's how cell culture people generally do the math and the cells don't care if it's 4.5% or 5%).
Filter sterilize.
Heat Inactivating Fetal Bovine Serum:
- Place a 500ml bottle from the -20C walk in freezer upstairs into the fridge or cold room and let it thaw over a couple of days or leave it to thaw on lab bench overnight. Make a control bottle containing water to heat alongside.
- Preheat a water bath to 55C
- Place both the FBS and control bottles into the 55C water bath. Check the temperature in the control bottle. Swirl bottles every 5-10 minutes and start timing when the temperature of the control bottle reaches 55C.
- Heat for 1 hour.
- In the sterile hood, aliquot the heat-inactivated FBS into 50ml tubes putting no more than 40mls per tube. (So tubes don't fracture when serum is refrozen)
- Put 50ml tubes into a styrofoam rack and freeze with the tubes upright (so caps stay on during freezing).
- Store long term in the ice chest in the walk-in -20C freezer.
|
Procedures Homepage | Solutions
http://research.biology.arizona.edu/mosquito/willott/ewlab.html
willott@u.arizona.edu
copyright 1999, willott
Last update July 25, 2000
Willott Lab Homepage
|