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Glycogen Determination from Tissues

38 bytes removed, 13:02, 19 June 2017
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==Protocol==
# Weight Weigh 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 mixing by gently tapping the vials to ensue the tissue dissolves completely. Make sure all the sample is initially immersed in KOH.
# Resuspend pellets in 200uL amyloglucosidase solution and incubate at 37C for 3h-O/N
# Quantify glucose using kit:
## Add 700 100 uL of Glucose Buffer Solution with Color Reagent buffer solution to a plastic cuvette. (each well, including wells for microplate add 100ul)standard curve and blank## Add 1-5 uL glucose standard (500mg/dL) for standard curve (for microplate add 1-5 ul of glucose standard 200mg/dL diluted 1:5)## Add 10 uL digested glycogen (for mice fasted more than 6 hours. samples, and use a 10X dilution for fed/short fast need lessrefed tissues (using either 5 or 2ul from the diluted sample)
## Mix and incubate at 37C for 5 min
## Measure absorbance at 505 nm
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