52
edits
Changes
m
Added step 2,clarified how to mix sample, added clarification to step 3 as to ensure all sample is immersed in KOH, added step 6, clarified the washing steps in order, clarified how to dry pellet
==Protocol==
# Weight out 30-90 mg tissue into a '''screw cap vial''' and record weights. Screw cap vials are really important or else the lids will pop off. # Turn on the heating block and set it to 95C (this can take up to 15 minutes to reach the desired temperature).# Add 300 uL 30% KOH and place on 95C heat block for 30 min with occasional mixingby gently tapping the vials to ensue the tissue dissolves completely. Make sure all the sample is initially immersed in KOH.
# Cool and then add 100 uL Sodium Sulfate and 800 uL Ethanol.
# Boil for 5 min.
# Turn on the incubator and set it to 37C.
# Centrifuge at 13 000 RPM for 5 min.
# Aspirate the solution leaving the pellet at the bottom of the vial.# Resuspend pellet in 200 uL water while making sure that all the glycogen dissolves in water, then add 400 uL ethanol. # Boil 5 min, spin 5 min and Repeat wash steps twice more(Wash Steps: Aspirate -> Resuspend with H20 and EtOH -> Boil -> Centrifuge -> Aspirate).# Dry pellet on the benchby leaving the vial cap open.
# Prepare amyloglucosidase solution by diluting the AG stock 100X into 50 mM Sodium Acetate, pH 4.8. Prepare enough for 200 uL per tube plus some extras
# Resuspend pellets in 200uL amyloglucosidase solution and incubate at 37C for 3h-O/N