Changes

* '''Homogenization Buffer''' (50 mM Tris pH 8, 5 mM EDTA, 30 mM Mannitol, PI inhibitor, can be made in bulk without the PI, PI added fresh) (to make 50 mL of homogenization buffer - 2.5 mL of 1M Tris pH 8, 500 uL of 0.5 M EDTA, 0.273 g Mannitol, fill up the rest with water)* 10M KOH(28.1g in 50 mL of water)

#Use 5 female or 8 Male flies Weigh out 30-50mg tissue (record weights for Assaynormalization) on dry ice into round bottom eppendorf tube (2 mL). 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 5ul KOH#Mix by inverting then transfewr sample to a new 1.5 mL tube. Place dirty ball bearing in ethanol (from Tween-20 stocklocated in the fume hood)#Heat samples in 70C waterbath 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 200 ul of supernatent in the bottom layer into a new microfuge tube#Spin @ 14000G Centrifuge again for 3 7-10 minutes@ 13000G#Transfer 150 ul of the bottom layer into the new tube (if there is enough of the bottom layer, transfer a total of 200 ul)#Let evaporate overnight at room temperature#Add '''(50ul )''' of '''Butanol Mixture''' and vortex. 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 80uL of TG solution glycerol reagent and 20uL of triglyceride reagent. Make extra and combine in a Falcon tube.##Aliquot '''100ul into a well of a 96 well plate'''#Incubate plate @ 37C for #For standards, add 0-5 and .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.##Pop any bubbles with tip before incubating.##Let sit for ~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.