TITLE: Total Starch (as Glucose) Quantification by NZYtech GOD-POD Method
AUTHORS: Lynn    Doran, Amanda  P. De Souza

[DESCRIPTION]
Total starch was previously extracted from plant tissue and enzymatically digested to D-Glucose.  This protocol is based on the NZYtech GOD-POD kit's colorimetric determination of D-Glucose and includes basic calculations to convert D-Glucose back to total starch in the original tissue. 

[BEFORE_START]
Extract and dry total starch pellet from plant tissue per Extraction of Non-Structural Carbohydrates (Total Soluble Sugars + Starch) in Plant Tissues.  Enzymatically digest starch to glucose per Total Starch Enzymatic Digestion.

[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, 2.5, 5, 7.5, 10, 12.5 ug. Include the 0 ug glucose/well on all plates.

Analyze samples in 3 technical replicates.

R2 of standard curve should be >0.995.   Typical standard deviation between technical sample replicates is 0.00 and 0.06 depending on precision of multi-channel or repeat pipettor used.  

This method is a modification of NZYTech Standard Protocol and NZYTech Alternative Procedures.

[STEPS]
SECTION: Prepare Glucose Standards
1. Prepare glucose standards in microcentrifuge by pipetting the appropriate amounts of 1 mg/mL Glucose standard and distilled water into each labeled tube.
 
    ABC1ug Glucose/50 ul-well (ug)Amount 1 mg/mL Glucose (ul)Amount distilled water (ul)2520180310401604156014052080120625100100
 
SECTION: Prepare Glucose Standards
2. Pipette 20 ul of each prepared glucose standard in triplicate into the assigned wells.


SECTION: Sample Preparation
3. Extract and digest total starch from plant tissue per Extraction of Non-Structural Carbohydrates (Total Soluble Sugars + Starch) in Plant Tissues. and Total Starch Enzymatic Digestion.
SECTION: Sample Preparation
4. Pipette 5-20 ul of each sample extract in triplicate into the assigned wells.  Record the amount of sample added.
 

SECTION: Assay
5. Prepare GOD-POD reagent per the instructions in the NZYtech GOD-POD protocol.  Follow shelf life and storage requirements as listed in the NZYtech GOD-POD protocol.
SECTION: Assay
6. Add 300 uL of prepared GOD-POD in each well using the multi-channel.
 

SECTION: Assay
7. Incubate the plates at 45°C for 20 min.
 

SECTION: Assay
8. Read the absorbance  at 510 nm on a UV-VIS spectrophotometer.

SECTION: Additional Assay Plates
9. 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
10. Include a blank, 0 ug glucose  standard (20 ul distilled water) on every plate.


SECTION: Basic Calculations
11. Normalize each assay plates absorbances to zero using the 0 ug glucose standard per plate.

SECTION: Basic Calculations
12. Calculate ug total starch 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
13. Divide the ug total starch by the ul of total sample extract loaded.
SECTION: Basic Calculations
14. Multiply the ug total starch per ul of total sample extract loaded by the total number of uls of buffer the total starch was re-suspended in.  If following the protocol, Extraction of Non-Structural Carbohydrates (Total Soluble Sugars + Starch) in Plant Tissues. and Total Starch Enzymatic Digestion. , as written the total starch was resuspended in 2000 uls of buffer.
SECTION: Basic Calculations
15. Divide the ug total starch per 2 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 Starch (as glucose) per mg plant tissue.