Changes

* '''Homogenization Buffer''' (50 mM TrispH 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)

* Sigma Triglyceride Assay Kit (Cat 337-BTR0100)

# Weigh out 20030-500mg 50mg tissue (record weight weights for normalization)# Homogenize with dounce homogenizer in on dry ice into round bottom eppendorf tube (2 mL '''). Add one stainless steel ball bearing.#Add 500ul Homogenization Buffer'''.# Remove 200 uL to a tube containing Homogenize with Qiagen Tissue Lyser for 3 minutes @ 25Hz for Liver/WAT or for 5 uL minutes @ 30Hz for muscle#Add 12.5ul KOH# Mix by invertingthen transfewr sample to a new 1.5 mL tube. Place dirty ball bearing in ethanol (located in the fume hood)# Add 800 uL 800ul '''Chloroform/Methanol Mixture'''# Vortex vigorously then sit at room temperature for 5 minminutes# Centrifuge 10min at 13 000 RPMfor 10 minutes @ 13000G# Take 180 uL Transfer 200 ul of the bottom layer into a fresh new tube.# Dry in fume hood overnight Centrifuge again for 7-10 minutes @ 13000G#Transfer 150 ul of the bottom layer into the new tube (or until completely dryif there is enough of the bottom layer, transfer a total of 200 ul)#Let evaporate overnight at room temperature# Add 50uL '''(50ul)''' of '''Butanol Mixture'''and vortex. See Suggested Volumes for your specific tissue.# Measure triglyceride levels using Sigma SIgma Diagnostic Kit , using 5 uL 5ul of sample.##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 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'''##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 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.