Changes

# Weigh out 30 mg tissue (record weight Use 5 female or 8 Male flies for normalization) on dry ice into round bottom eppendorf tube. Add one stainless steel ball bearing. # Add 500 uL Homogenization Buffer.Assay# Homogenize with qiagen tissue lyser flies (3 minutes at 25 Hz for Liver/WAT or 5 min at 30 Hz for muscle@ 40Hz) in 1mL of .05% Tween (from Tween-20 stock)# Add 12.Heat samples in 70C waterbath for 5 uL KOHminutes# Mix by inverting# Add 800 uL '''Chloroform/Methanol Mixture'''# Vortex vigorously then sit at room temperature Spin samples @ 5000G for 5 min1 minute# Centrifuge 10min at 13 000 RPM# Take the bottom layer into a fresh labelled Transfer 500ul of supernatent in new microfuge tube. # Dry in fume hood overnight (or until completely dry)# If absorbance is going to be measured by cuvette, use non-bolded values. If you are using a plate reader use bolded values.Spin @ 14000G for 3 minutes# Add 500uL '''(50uL)''' of '''Butanol Mixture'''. See [[#Suggested Volumes | Suggested Volumes]] for your specific tissue# Measure triglyceride levels using Sigma Diagnostic Kit using 5 uL 50ul of sample:## Resuspend triglyceride and glycerol reagent with water if necessary.## Calculate how many samples you have (samples + buffer blank + 6 standard curve values).## Prepare reagent, you need 560 uL '''(80uL)''' to 200ul of glycerol reagent and 140 uL '''(20uL)''' of triglyceride reagent. Make a bit extra and combine TG solution in a falcon tube.## Aliquot 700 uL into a cuvette or '''100 uL into a well of a 96 well plate'''.## For standards add 0-5 uL of glycerol standard.## Add 5 uL of resuspended lipid to each well to start (also make a 5uL blank of the butanol mixture) and mix.## Let sit Incubate plate @ 37C for ~30 min at room temperature (or 5 min at 37C if you are in a hurry). If using > 10 uL of butanol mixture the solution may be cloudy, let it settle and it should become more clear.mins## Measure 540nm absorbance at 540 nm.## If any samples are A540<0.1 or above the 5uL standard A540 then repeat with more or less lipid as required.