TITLE: Total Soluble Sugar Quantification from Ethanolic Plant Extracts        
AUTHORS: Lynn    Doran, Amanda  P. De Souza

[DESCRIPTION]
Quantification of total soluble sugars (as glucose) in plant tissue extracts via the sulfuric phenol method adapted for 96 well plates.

[BEFORE_START]
Extract and purify total soluble sugars from plant tissue per Extraction of Non-Structural Carbohydrates (Total Soluble Sugars + Starch) in Plant Tissues.

[GUIDELINES]
A standard curve is necessary for each day of measurement. If you are doing multiple plates in the same day with turning off the plate reader, it is okay have the standard curve only in one plate. For the standard curve, use the following amounts of glucose (Glc): 0, 5, 10, 15,20, 25 ug. Include the 0 ug glucose/well on all plates.

Analyze samples in 3 technical replicates.

R2 of standard curve should be >0.990.   Typical standard deviation between technical sample replicates is 0.10 and 0.30 depending on precision of multi-channel or repeat pipettor used.  

[STEPS]
SECTION: Glucose standard preparation
1. Prepare glucose standards in microcentrifuge by pipetting the appropriate amounts of 1 mg/mL Glucose standard and distilled water into each labeled tube.

 

SECTION: Glucose standard preparation
2. Pipette 50 ul of each prepared glucose standard in triplicate into the assigned wells.
4. Pipette 10-30 ul of each sample extract in triplicate into the assigned wells.  Record the amount of sample added.
 

5. Add distilled water to bring the total volume of each well to 50 ul.  For example, if 10 ul of sample was used add 40 ul of distilled water.
SECTION: Sample preparation
3. Extract and purify total soluble sugars from plant tissue per Extraction of Non-Structural Carbohydrates (Total Soluble Sugars + Starch) in Plant Tissues.
SECTION: Assay
6. Move the plate to the chemical fume hood.
SECTION: Assay
7. Inside the hood, add 150uL of sulfuric acid to each well.  Try to minimize the time between addition to first well and final well. 
 

 

SECTION: Assay
8. Immediately after the addition of sulfuric acid, add 30uL of phenol 5% in each well.  Try to minimize the time between addition to first well and final well. 
 

 

SECTION: Assay
9. Incubate the plate by floating in a 90 °Cwater bath for 5 min.
SECTION: Assay
10. Place plate on ice bath to cool until cool to the touch.
SECTION: Assay
11. Once the plate is cool, read the absorbance at 490 nm on a UV-VIS spectrophotometer.
SECTION: Additional Assay Plates
12. Do not shutoff the spectrophotometer lamp between plates.  If the lamp remains on continuously, only one glucose standard curve is needed.  If the lamp is shut off, a standard curve will need to be included.
SECTION: Additional Assay Plates
13. Include a blank, 0 ug glucose  standard (50 ul distilled water) on every plate.
SECTION: Basic Calculations
14. Normalize each assay plates absorbances to zero using the 0 ug glucose standard per plate.
SECTION: Basic Calculations
15. Calculate ug total soluble sugar as glucose for each sample using the averaged  normalized standard curve absorbances for technical replicates and the averaged normalized sample absorbances for technical replicates. 
SECTION: Basic Calculations
16. Divide the ug total soluble sugar by the ul of total sample extract loaded.
SECTION: Basic Calculations
17. Multiple the ug total soluble sugar per ul of total sample extract loaded by the total number of uls of distilled water the total soluble sugars were re-suspended in.  If following the protocol, Extraction of Non-Structural Carbohydrates (Total Soluble Sugars + Starch) in Plant Tissues., as written the total soluble sugar was resuspended in 1000 uls of distilled water.
SECTION: Basic Calculations
18. Divide the ug total soluble sugar per 1 mL extract by the initial weight of ground tissue used in the ethanolic extraction (Step 1 of Extraction of Non-Structural Carbohydrates (Total Soluble Sugars + Starch) in Plant Tissues.)  The final value reported will be ug Total Soluble Sugars (as glucose) per mg plant tissue.