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)

# Weigh out 20030-500mg 50mg tissue (record weight weights for normalization). You can use as low as 30 mg tissue if necessary by reducing the lysis volume on dry ice into round bottom eppendorf tube (must be at least 500 uL) or increasing the amount of the lower (chloroform) layer removed up to 500 ul(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 Transfer 200 uL ul of the bottom layer into a fresh new tube. See [[#Suggested Volumes | Suggested Volumes]] Centrifuge again for your specific tissue7-10 minutes @ 13000G# Dry in fume hood overnight 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)# If absorbance is going to be measured by cuvette, use non-bolded values. If you are using a plate reader use bolded values.Let evaporate overnight at room temperature# Add 50uL '''(500uL50ul)''' of '''Butanol Mixture'''and vortex. See [[#Suggested Volumes | 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 > 10 uL 10ul of butanol mixture mix the solution may be cloudy, let . 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. ===Suggested Volumes===This is based on using a 96 well plate to measure final concentrations.{| border="1"|-! Tissue/Condition !! Lysis Volume !! Chloroform Volume !! Resuspension Volume !! Assay Volume |-| Liver || 1 mL || 200 uL || 500 uL || 5 uL |-| Muscle || 1 mL || 200 uL || 50 uL || 5 uL |}