Glycogen Determination from Tissues: Difference between revisions

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==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.

# 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.

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# Resuspend pellets in 200uL amyloglucosidase solution and incubate at 37C for 3h-O/N

# Quantify glucose using kit:

## Add ~~700~~ uL Glucose ~~Buffer Solution with Color Reagent~~ to ~~a plastic cuvette. (~~for ~~microplate add 100ul)~~

## Add 100 uL of Glucose buffer solution to each well, including wells for 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 1-5 ul of glucose standard 200mg/dL diluted 1:5

## Add 10 uL digested glycogen (for ~~mice~~ fasted ~~more than 6 hours.~~ for ~~fed/short fast need less~~)

## Add 10 uL digested glycogen for fasted samples, and use a 10X dilution for refed tissues (using either 5 or 2ul from the diluted sample)

## Mix and incubate at 37C for 5 min

## Measure absorbance at 505 nm

@@ Line 15: / Line 15: @@
 ==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.
+# 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.
@@ Line 29: / Line 29: @@
 # Resuspend pellets in 200uL amyloglucosidase solution and incubate at 37C for 3h-O/N
 # Quantify glucose using kit:
-## Add 700 uL Glucose Buffer Solution with Color Reagent to a plastic cuvette. (for microplate add 100ul)
+## Add 100 uL of Glucose buffer solution to each well, including wells for 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 1-5 ul of glucose standard 200mg/dL diluted 1:5
-## Add 10 uL digested glycogen (for mice fasted more than 6 hours. for fed/short fast need less)
+## Add 10 uL digested glycogen for fasted samples, and use a 10X dilution for refed tissues (using either 5 or 2ul from the diluted sample)
 ## Mix and incubate at 37C for 5 min
 ## Measure absorbance at 505 nm