Changes

#Use 5 female or 8 Male flies Weigh out 30mg tissue (record weights for Assaynormalization) on dry ice into round bottom eppendorf tube. Add one stainless steel ball bearing.#Add 500ul Homogenization Buffer#Homogenize flies (with Qiagen Tissue Lyser for 3 min minutes @ 40Hz) in 1mL of 25Hz for Liver/WAT or for 5 minutes @ 30Hz for muscle#Add 12.05% Tween (from Tween-20 stock)5ul KOH#Heat samples in 70C waterbath Mix by inverting #Add 800ul '''Chloroform/Methanol Mixture'''#Vortex vigorously then sit at room temperature for 5 minutes#Spin samples @ 5000G Centrifuge for 1 minute10 minutes @ 13000G#Transfer 500ul of supernatent in the bottom layer into a new microfuge tube#Spin @ 14000G for 3 minutesLet evaporate overnight at room temperature#If absorbance is going to be measured by cuvette, use non-bolded values. If using a plate reader, used bolded values.#Add 500ul '''(50ul )''' of '''Butanol Mixture'''. See Suggested Volumes for your specific tissue.#Measure triglyceride levels using SIgma Diagnostic Kit, using 5ul of sample to 200ul .##Resuspend triglyceride and glycerol reagent with water if necessary##Calculate how many sample you have (samples + blank + standard curve)##Prepare reagent. You need 560ul '''(80ul)''' of TG solution glycerol reagent and 140ul '''(20ul)''' of triglyceride reagent. Make extra and combine in a Falcon tube.##Aliquot 700ul into a cuvette or '''100ul into a well of a 96 well plate'''#Incubate plate @ 37C #For standars, add 0-5ul of glycerol standard##Add 5ul of resuspended lipid to each well to start (also make a 5ul blank of the butanol mixture) and mix.##Let sit for 5 ~30 mins@ room temperature (or 5mins @ 37C if you are in a hurry). If using >10ul of butanol mix the solution may be cloudy. Let it settle and it should become more clear.##Measure 540nm absorbance@ 540nm##If any samples are A540<0.1 or above the 5ul standard the A540, then repeat with more or less lipid as required.