915
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Changes
added categories
===DAY1===
# Grow 10 ml starter cultures at 24C 24C in shaker in the appropriate media
===DAY2===
# Once the cells reach an OD600= 0.600, we will use them to inoculate a culture that needs to grow for exactly 12 hours (10:00 PM to 10:00 AM tends to work well).
# Prepare appropriate Inositol-free media
# At around 9:30 PM (If you are culturing from 10 to 10) begin preparations for inoculating the cells and for working with radioactivity. Get absorbent paper for bench space and bag for radioactive waste
# For each sample, prepare three 50ml falcon tubes
# Resuspend the cells in 875 l inositol-free media (2 OD/ml)
# At 10:00 PM inoculate each of the three 50 ml Falcon tubes with the appropriate amount of each cell type based on the following considerations
## The amount for inoculation will vary based on the growth rate of each cell strain (Wild type cells need about 60 luL)
## We want the cultures to grow to an OD600=0.600 in 12 hours
# Add 50 ul of 1uCi/uL myo-H3-inositol to the No Salt and Salt tubes (not to the growth control tubes)
===DAY 4===
# Thaw the samples on the bench for 10 minutes
# Resuspend each sample with 300 l uL ddH2O
# Leave at room temp for 30 minutes
# Resuspend each sample again
# The amount of sample to use in the HPLC can vary depending on intensity of signal, but a good starting point is 15 uL of sample in 35 uL ddH2O (total 50 uL)
# Load the entire 50 uL on the HPLC
[[Category: Lipid Analysis]]
[[Category: Inositol Lipids]]
[[Category: Yeast]]
