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). You can use less tissue if necessary by reducing the lysis volume on dry ice into round bottom eppendorf tube (must be greater than 500 uL) or increasing the amount of chloroform layer removed (normally 180 uL2 mL). Add one stainless steel ball bearing.# Homogenize with dounce homogenizer in 2 mL '''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 (or until completely dry)Centrifuge again for 7-10 minutes @ 13000G# If absorbance Transfer 150 ul of the bottom layer into the new tube (if there is going to be measured by cuvetteenough of the bottom layer, use non-bolded values. If you are using transfer a plate reader use bolded values.total of 200 ul)#Let evaporate overnight at room temperature# Add 50uL '''(500uL50ul)''' 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 samples sample you have (samples + buffer blank + 6 standard curve values).## Prepare reagent, you . You need 560 uL '''(80uL)''' of glycerol reagent and 140 uL '''(20uL)''' of triglyceride reagent. Make a bit extra and combine in a falcon Falcon tube.## Aliquot 700 uL into a cuvette or '''100 uL 100ul into a well of a 96 well plate'''.## For standards , add 0-5 uL and .5ul of glycerol standard '''(or of a 1/10 dilution of the glycerol standard)'''.## Add 5 uL 5ul of resuspended lipid to each well to start (also make a 5uL 5ul blank of the butanol mixture) and mix.##Pop any bubbles with tip before incubating.## Let sit for ~30 min at mins @ room temperature (or 5 min at 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 at 540 nm.@ 540nm## If any samples are A540<0.1 or above the 5uL 5ul standard the A540 , then repeat with more or less lipid as required.