Inositol Labelling of Yeast Cells: Difference between revisions
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===DAY1=== | ===DAY1=== | ||
# Grow 10 ml starter cultures at | # Grow 10 ml starter cultures at 24C in shaker in the appropriate media | ||
===DAY2=== | ===DAY2=== | ||
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# 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). | # 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 | # 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 | # 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 | # For each sample, prepare three 50ml falcon tubes | ||
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# Resuspend the cells in 875 l inositol-free media (2 OD/ml) | # 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 | # 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 | ## The amount for inoculation will vary based on the growth rate of each cell strain (Wild type cells need about 60 uL) | ||
## We want the cultures to grow to an OD600=0.600 in 12 hours | ## 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) | # Add 50 ul of 1uCi/uL myo-H3-inositol to the No Salt and Salt tubes (not to the growth control tubes) | ||
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===DAY 4=== | ===DAY 4=== | ||
# Thaw the samples on the bench for 10 minutes | # Thaw the samples on the bench for 10 minutes | ||
# Resuspend each sample with 300 | # Resuspend each sample with 300 uL ddH2O | ||
# Leave at room temp for 30 minutes | # Leave at room temp for 30 minutes | ||
# Resuspend each sample again | # Resuspend each sample again | ||
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# 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) | # 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 | # Load the entire 50 uL on the HPLC | ||
[[Category: Lipid Analysis]] | |||
[[Category: Inositol Lipids]] | |||
[[Category: Yeast]] | |||