34
edits
Changes
updated volumes and lysis procedure
==Protocol==
# Weigh out 200-500mg 30 mg tissue (record weight for normalization)on dry ice into round bottom eppendorf tube. You can use as low as 30 mg tissue if necessary by reducing the lysis volume (must be at least Add one stainless steel ball bearing. # Add 500 uL) or increasing the amount of the lower (chloroform) layer removed up to 500 ul(normally 180 uL)Homogenization Buffer.# Homogenize with dounce homogenizer in 2 mL '''Homogenization Buffer'''qiagen tissue lyser 3 minutes at 25 Hz for Liver/WAT or 5 min at 30 Hz for muscle.# Remove 200 uL to a tube containing Add 12.5 uL KOH
# Mix by inverting
# Add 800 uL '''Chloroform/Methanol Mixture'''
# Vortex vigorously then sit at room temperature for 5 min
# Centrifuge 10min at 13 000 RPM
# Take 200 uL of the bottom layer into a fresh labelled tube. See [[#Suggested Volumes | Suggested Volumes]] for your specific tissue
# Dry in fume hood overnight (or until completely dry)
# If absorbance is going to be measured by cuvette, use non-bolded values. If you are using a plate reader use bolded values.
## Measure absorbance at 540 nm.
## If any samples are A540<0.1 or above the 5uL standard A540 then repeat with more or less lipid as required.
