| Usage |
1. Self-brought consumables:
Microplate reader or visible spectrophotometer (capable of measuring 620 nm Absorbance at)
96 Well plate or micro glass cuvette, adjustable pipette gun and tip
Incubator, water bath, centrifuge
Deionized water, concentrated sulfuric acid
Mortar, sealing film
2. Reagent preparation:
Extraction Buffer ： Ready-to-use; Before use, equilibrate to room temperature; 4℃ Save.
Reagent I ： Ready-to-use; Before use, equilibrate to room temperature; 4℃ Save.
Reagent II ： Prepare before use; join 5.25 mL After distilling water, add slowly 29.75 mL Concentrated sulfuric acid, constantly stirring, fully dissolving for later use; 4℃ Keeps up to one week protected from light.
Note: Reagent I Has a pungent smell, Reagent II It is toxic and concentrated sulfuric acid is highly corrosive. It is recommended to conduct experiments in fume hoods.
Standard ： Prepare before use; join 1 mL Dissolve it with deionized water, that is, 10 mg/mL Glucose standard solution, 4℃ Savable 2 Zhou.
Standard preparation: Using 10 mg/mL Standard solution, further diluted to standard as shown in the table below:
| Serial number |
Standard Volume |
Volume of deionized water ( µL ） |
Concentration ( mg/mL ） |
| Std.1 |
200 µL 10 mg/mL Standard |
1800 |
1 |
| Std.2 |
400 µL of Std.1 (1 mg/mL) |
600 |
0.4 |
| Std.3 |
200 µL of Std.1 (1 mg/mL) |
800 |
0.2 |
| Std.4 |
100 µL of Std.1 (1 mg/mL) |
900 |
0.1 |
| Std.5 |
50 µL of Std.1 (1 mg/mL) |
950 |
0.05 |
| Std.6 |
40 µL of Std.1 (1 mg/mL) |
960 |
0.04 |
| Std.7 |
30 µL of Std.1 (1 mg/mL) |
970 |
0.03 |
| Std.8 |
20 µL of Std.1 (1 mg/mL) |
980 |
0.02 |
| Blank |
0 |
1000 |
0 |
Note: Every experiment should be tested for standard products and the standard song should be made; The diluted standard solution is unstable and must be prepared at 4 Use within hours.
3. Sample preparation:
Note: 1 、 It is recommended to use fresh samples. If the experiment is not carried out immediately, the samples can be found at -80℃ Keep for a month. During measurement, the temperature and time of thawing should be controlled. Room temperature environment When thawing, it needs to be 4 h The sample thawing was completed within. 2 Some reagents are highly corrosive and must be protected during operation.
1 Plant tissue 37℃ After oven drying or natural air drying, weigh about 0.03 g The sample was well ground in a mortar and added 1 mL Extraction Buffer , rapid homogenization followed by transfer to 2 mL EP In the tube (cap tightly and wrap the nozzle with sealing film) 80℃ Water bath extraction 30 min ， 3,000 g ， 25℃ Centrifugation 5 min , discard the supernatant and leave the precipitate.
2 Adding to the precipitate 0.5 mL Deionized water, put in 95℃ Gelatinization in water bath 15 min (Cap tightly and wrap the nozzle with sealing film).
3 After cooling, add 0.35 mL Reagent I , put in 95℃ Extraction in water bath 15 min (Cap tightly and wrap the nozzle with sealing film), oscillating during 3-5 Times.
4 After cooling, add 0.85 mL Deionized water, mixing evenly, 3,000 g ， 25℃ Centrifugation 10 min , take the supernatant to be tested.
4. Experimental steps:
1 , microplate reader or visible spectrophotometer preheat 30 min , adjust the wavelength to 620 nm 。 The visible spectrophotometer is zeroed with deionized water.
2 , operation table (the following operations are in 1.5 mL EP Conducted in tube):
| Reagent |
Determination tube ( μL ） |
Standard tube ( μL ） |
Blank tube ( μL ） |
| Sample supernatant |
50 |
0 |
0 |
| Standard |
0 |
50 |
0 |
| Deionized water |
0 |
0 |
50 |
| Reagent II |
250 |
250 |
250 |
3 After mixing well, put in 95℃ Water bath 15 min (Cover tightly and wrap the nozzle with sealing film), naturally cool to room temperature, mix well, take 200 μL To micro cuvettes or 96 In the well plate, determine 620 nm Absorbance, respectively recorded as A determination 、 A Criteria 、 A Blank , calculation ΔA determination =A determination -A Blank 、 ΔA Criteria =A Criteria -A Blank 。 Note: Standard curve and blank tube only need to do 1-2 Times. It is recommended to select before the experiment 2-3 Do pre-experiments with samples with large expected differences, if A determination Greater than 1.0 , sample supernatant Available extracts (as per 0.35 mL Reagent I ： 1.35 mL Proportion of deionized water, prepared as needed) for further dilution, and the calculation result is multiplied by the dilution factor, if A measure Fixed Less than A Blank The sample size can be appropriately increased.
5. Result calculation:
Note: The calculation formulas we provide you include derivation process calculation formulas and concise calculation formulas. Both are perfectly equal. It is recommended that the bold concise calculation formula be the final Calculation formula.
1 , drawing of standard curve ;
Taking the standard solution concentration as x Shaft, ΔA Criteria For y Axis, draw the standard curve, and get the standard equation y=kx+b , will ΔA determination Brought into the equation to get x (mg/mL) 。
2 Calculation of starch content
Starch content (mg/g Dry weight )=x×V Sample ÷(W×V Sample ÷V Sample total )÷1.11×N=x×1.53÷w×N
V Sample : sample volume added into the reaction system, 0.05 mL ； V Sample total : join Extraction Buffer Volume, 1.7 mL ； W : sample quality, g ； 1.11 : is the constant of converting the glucose content measured by this method into starch content, that is 111 μg The color of glucose with anthrone reagent is equivalent to 100 μg The color of starch displayed with an anthrone reagent; N : Sample dilution factor.
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