915
edits
Changes
fixed some formatting errors
5 ml 10 ml
1.34 ml 2.68 ml 40% methylamine
0.805 ml 1.61 ml ddH2O
===n-Butanol-ethyl ether-formate (20:4:1) (make fresh each time)===
10 samples
5 ml n-Butanol (1-Butanol)
250 l formic acid ethyl ester
===Inositol-Free Media===
100 ml (Enough for 8 samples)
10 ml 10X Amino Acid Stock
1 ml 100X Vitamin Stock 1 ml 100X Trace Elements
5 ml 40% Glucose (When adding back amino acids Use: 1080 l Uracil (2mg/ml) ;
53 ml ddH2O 400 l uL Tryptophan (10 mg/ml); 864 l uL Leucine ; 216 l uL Histidine) ==Protocol=====DAY1===# Grow 10 ml starter cultures at 24C in shaker in the appropriate media ===DAY2===# Check the OD600 of starter cultures# Back dilute the cultures appropriately based on the following considerations -- We want a final OD600= 0.600# 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## Label the tubes appropriately (i.e. Growth Control, No Salt, 10 min Salt)## Put 5 ml inositol-free media in each# Pellet 1.75 OD600 of each cell type in a 1.7 ml microcentrifuge tube (14,000 RPM for 1 minute)# Aspirate the supernatant# Wash the cells one time in 1 ml inositol-free media (resuspend cells, spin at 14,000RPM for 1 min, and aspirate supernatant)# 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 l)## 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)# Gently swirl the tubes to disperse the radioactivity evenly# The lids to the falcon tubes should be loose, but taped to prevent contamination ===DAY 3===# Before the 12 hour time point prepare 15 ml 4.5% Perchloric Acid and Chill on ice (14.04 ml dH2O and 965 uL 70% Perchloric Acid)# Prepare a 2.0 ml screw cap tube for each sample by ## Filling it half full with 0.5mm zirconia beads (Biospec Products Inc. Cat # 11079105z www.biospec.com)## Adding 470 uL 4.5% Perchloric Acid## Numbering it (and noting which sample goes with each number)# At 12 hours of culturing, measure the OD600 of the growth control tubes to assure proper growth## Discard the remaining growth control culture## Close the lids on the radioactive samples and pellet the cells (3,000 RPM for 3 min)# Being aware of radioactivity, remove the supernatants with 10 ml serological pipettes and place in H3 liquid waste# Wash each cell pellet with 3 ml inositol-free media (resuspend cells, spin at 3,000 RPM for 3 min)# Being aware of radioactivity, remove the supernatants with 10 ml serological pipettes and place in H3 liquid waste# Using the residual liquid in the tubes, resuspend the cells and measure the volume (this should be less than 100 uL)# Bring the volume of the cells to 100 uL using inositol-free media# Add an additional 100 uL inositol-free media to the “No Salt” tubes# The “Salt” tubes will have salt added for varying amounts of time…be sure to have a timer ready# Add 100 uL inositol-free media containing 1.8 M NaCl to the “Salt” tubes# Incubate the cells for the appropriate time interval with gentle swirling every 3-4 minutes# At precisely the correct time interval stop the reaction by adding 800 uL 4.5% Perchloric Acid# Place the tubes on ice# Being certain not to overflow the tubes, transfer the cell mixture to the appropriately numbered 2 ml screw cap tube (prepared earlier with zirconia beads and perchloric acid)# This should bring the tube to full volume# Lyse the cells by placing each tube into the “Bead Beater” and shake for 2 minutes at high speed# Immediately place the cells on ice for 2 minutes# Repeat this process two more times for a total lysis time of 6 minutes# Using a P1000 transfer the lysed cells to labeled 1.5 ml microcentrifuge tubes. After removing the lysed cells above the beads, dig the tip into the beads and suck up the remaining liquid. Try to prevent beads from going into the 1.5 ml tubes by placing the tip against the edge of the tube while ejecting# Centrifuge the lysed cells at 14,000 RPM for 10 minutes at 4C# Remove and discard the supernatant in liquid H3 waste using a P1000# Resuspend each pellet with 1ml 100mM EDTA## If beads have snuck through, try to remove them at this point## Centrifuge 14,000 RPM for 10 minutes at 4C## Remove and discard the supernatant in liquid H3 waste using a P1000## Resuspend the pellets in 50 uL ddH2O# Transfer each sample to a 5 ml glass vial. SAVE the 1.5 ml tubes for washing in the next step# Add 1ml methylamine reagent to each sample. Use the methylamine reagent to wash the appropriate 1.5 ml tube for each sample# Tighten the lids on the 5 ml glass vials and heat the vials at 55C for 1 hour# Allow the tubes to cool to room temp (*approximately 5 minutes)# Transfer the supernatant to labeled 1.5 ml microcentrifuge tubes# Dry the samples in the speed vac for about 3 hours# Meanwhile, begin the clean up process## Empty liquid H3 waste into appropriate container## Catalogue in the Radiation Isotope Log book the amount of radiation you used## Clean the pipettes with “Lift Away”## Clean centrifuges with “Lift Away”# Store your samples at -20C until tomorrow# Clean the speed vac with “Lift Away” ===DAY 4===# Thaw the samples on the bench for 10 minutes# Resuspend each sample with 300 l ddH2O# Leave at room temp for 30 minutes# Resuspend each sample again# Vortex each sample for about 30 seconds# Centrifuge at 14,000 RPM for 2 min at room temp# Transfer supernatant to new 1.5ml labeled tube# Add 300 uL butyl-ethyl ether-formate solution# Vortex each tube for 15 seconds# Centrifuge the samples 14,000 RPM for 2 min# Transfer the bottom layer (aqueous phase) to a new 1.5 ml tube using a P200# Add another 300 uL butyl-ethyl ether-formate solution## Repeat vortex and centrifugation steps as above## Transfer the bottom layer (aqueous phase) to a new 1.5 ml tube using a P200## Speed vac the samples (to dry) for about 1.5 hours## Place the samples at -20C until loading on HPLC (Be sure to clean Speed Vac) ===HPLC Sample Preparation===# Thaw the samples at room temp for about 5 minutes# Resuspend the sample in 20 uL ddH2O# Let sit at room temp for 5 minutes# Centrifuge the sample at 14,000 RPM for 1 min (to pellet any particulate matter)# 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