Standard Operating Procedure
Title: Sugar Assay
Department: Agronomy
Created by: Andrea Rouse
Laboratory: Crop Production & Physiology
Supervisor: Mark Westgate
Lab Supervisor: Maria Hartt
Date approved:
Procedure Overview: Determination of soluble sugar content in maize samples
Equipment and reagents necessary:
Reagents:
80% Ethanol (15 ml per sample)
Concentrated Trichloroacetic acid (a few drops per sample)
0.1N Hydrochloric acid (a few drops per sample)
5N Potassium Hydroxide (a few drops per sample)
Dinitrosalicylic acid (10g per liter of stock solution)
Sodium sulfite (0.5g per liter of stock solution)
Sodium hydroxide (10g per liter of stock solution)
Potassium sodium tartrate solution 40% (1ml per sample)
Equipment:
Balance
Weigh boat
Spatula
Conical centrifuge tubes, 50ml (1 per sample)
Pipettes, 5 ml, 1 ml & 20 ul & tips
Conical centrifuge tubes, 15ml (3 per sample)
Graduated cylinder
Heat block
Centrifuge
Water bath
Spectrophotometer (w/capability of reading 575nm wavelength)
Cuvettes (1 per sample)
Procedure:
1. Using balance, weigh 100mg of dried stalk tissue and place in a 15 mL conical
tube
2. Pipette 5ml of 80% Ethanol into tube; cap
3. In a heating block, heat tube at 60oC for 20 minutes
4. Remove tube from block and centrifuge at 5500g for 10 minutes at room temp
5. Transfer supernatant to a properly labeled, clean 50ml conical tube
6. Repeat from Step 2 for a total of 3 ethanol extractions.
supernatant in the 50 mL tube from step 5
Combine all the
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7. Precipitate proteins by adding a few drops of concentrated Trichloroacetic acid
(TCA) to tube containing the supernatant using a pipette (a typical drop is
approximately 20ul); cap tube
8. Centrifuge tube at 5500g for 10 minutes to pellet the proteins.
supernatant and place into a clean 15ml conical tube with cap
Retain the
9. Hydrolyze sucrose by adding a few drops of 0.1N Hydrochloric acid (HCl) to tube
containing the supernatant using a pipette
10. Place 3ml of above sugar solution in a clean 15mL conical tube and add 3ml
Dinitrosalicylic acid reagent solution (1% DNS: 10g Dinitrosalicylic acid, 0.5g
sodium sulfite, 10g sodium hydroxide, water to 1 liter). Tightly cap the tube or use
parafilm to eliminate evaporation
11. Heat the tube containing the sugar/DNS mixture in a 90oC heating block for 5-15
minutes to develop red-brown color
12. Add 1ml of 40% potassium sodium tartrate (Rochelle salt) solution to stabilize the
color
13. Cool sample to room temperature in a cold water bath
14. Transfer sample to a cuvette. Record absorbance using a spectrophotometer at
575nm. Compare absorbance to a glucose standard curve using the same
procedure
15. Place all remaining samples in properly tagged waste container for EH&S pick up.
Clean all work areas and lab ware.
Personal Protective Equipment / Engineering Controls:
Gloves
Face mask when using Dinitrosalicylic acid in dry form
Appropriate shoes
Goggles
Lab coat
Safety shower and eye wash station
Hazard Controls & Storage Precautions:
3,5-Dinitrosalicylic acid:
Ethanol:
Hydrochloric Acid:
Potassium Hydroxide:
Potassium Sodium Tartrate (Rochelle Salt):
Sodium Hydroxide:
Sodium Sulfite Anhydrous:
Trichloroacetic acid:
Waste Disposal & Decontamination Procedures:
3,5-Dinitrosalicylic acid:
Ethanol:
Hydrochloric acid:
Potassium Hydroxide:
Potassium Sodium Tartrate (Rochelle Salt):
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Sodium Hydroxide:
Sodium Sulfite Anhydrous:
Trichloroacetic acid:
Health & Safety Info for Required Reagents:
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Chemical name
3,5-Dinitrosalicylic
acid
Ethanol
Hydrochloric Acid
Potassium Sodium
Tartrate
Potassium
Hydroxide
Sodium Hydroxide
Sodium sulfite
Trichloroacetic acid
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Incompatibilities
 The above summary consists of guidelines for proper handling &
disposal of chemicals used in this procedure. You must read complete
MSDS(s) for more specific information before starting this procedure.
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