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Dubois Total Soluble Sugar Protocol

The document outlines the Phenol-Sulfuric Acid Method for estimating total soluble sugars, detailing materials, reagents, and a step-by-step protocol for sample preparation, reaction setup, incubation, and measurement. It emphasizes the importance of rapid sulfuric acid addition and the use of glass test tubes for accurate results. The method includes calculations for determining sugar concentration based on absorbance measurements against a glucose standard curve.

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423 views2 pages

Dubois Total Soluble Sugar Protocol

The document outlines the Phenol-Sulfuric Acid Method for estimating total soluble sugars, detailing materials, reagents, and a step-by-step protocol for sample preparation, reaction setup, incubation, and measurement. It emphasizes the importance of rapid sulfuric acid addition and the use of glass test tubes for accurate results. The method includes calculations for determining sugar concentration based on absorbance measurements against a glucose standard curve.

Uploaded by

Niall's 5 sexy potatoes
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© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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Dubois et al.

(1956) Phenol-Sulfuric Acid Method for Total Soluble Sugar Estimation

Materials and Reagents:

1. Sample extract (plant tissue, microbial culture supernatant, etc.)


2. 5% Phenol solution (w/v in distilled water)
3. Concentrated Sulfuric Acid (H2SO4, 95-98%)
4. Glucose standard (1 mg/mL stock solution in distilled water)
5. Distilled water
6. Pipettes and tips
7. Glass test tubes (preferably heat-resistant)
8. Spectrophotometer (490 nm, optional 480-500 nm range acceptable)

Protocol:

1. Preparation of Standards and Samples:


- Glucose standard curve: Prepare glucose solutions in the range of 10-100 ug/mL using distilled water.
- Sample preparation:
- Weigh 0.1-0.5 g of plant tissue and homogenize in 5-10 mL of distilled water.
- Heat at 80-90 degreesC for 10 min, cool, and centrifuge at 5000 rpm for 10 min.
- Collect the supernatant for sugar analysis.

2. Reaction Setup:
- Take 1 mL of sample or standard into a glass test tube.
- Add 0.05 mL (50 uL) of 5% phenol solution.
- Quickly add 5 mL of concentrated sulfuric acid (H2SO4) directly into the tube.
- Mix immediately by vortexing or shaking.

3. Incubation:
- Allow the reaction to proceed at room temperature (25-30 degrees C) for 30 minutes.
- The solution will develop an orange-yellow color, indicating sugar presence.

4. Measurement:
- Measure the absorbance at 490 nm using a spectrophotometer against a blank (distilled water + phenol +
- Prepare a standard curve using glucose solutions.
- Calculate the sugar concentration in unknown samples using the standard curve equation.

Calculations:

C_sample = (A_sample / A_standard) x C_standard


where:
- A_sample = Absorbance of the sample
- A_standard = Absorbance of the known glucose concentration
- C_standard = Concentration of the glucose standard

Notes & Precautions:

- Rapid addition of sulfuric acid is crucial for consistent results.


- Perform the assay in glass tubes only as sulfuric acid can damage plastic.
- Sulfuric acid generates heat upon mixing, so handle with care.
- Ensure proper linear response of the standard curve before sample calculations.

Reference:

Dubois, M., Gilles, K. A., Hamilton, J. K., Rebers, P. A., & Smith, F. (1956). Colorimetric method for
determination of sugars and related substances. Analytical Chemistry, 28(3), 350-356.
https://doi.org/10.1021/ac60111a017

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