IGEM:IMPERIAL/2009/M2/Assays/glu1

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Glucose Assay Kit

  • James Field 07:04, 12 August 2009 (EDT): According to their website, their UK distributor is:

UNITED KINGDOM CAMBRIDGE BIOSCIENCES, 24-25 Signet Court, New Market Road, Cambridge CB5 8LA, UK Tel: +1223 316855; Fax: +1223 360732; Email: tech@bioscience.co.uk

If we go for this kit, we need to send them an email and confirm the UK price and delivery time, and if they have it in stock.


(Catalog #K606-100; 100 assays; Store at -20°C.)

specifications

I. Introduction:

Since this assay uses fluorescence, it might be worth taking a sample from the culture and filtering (minsart) it before taking the reading. This would remove any E.coli cells and ensuring that GFP did not mess with the fluorescence read-out.


Glucose (C6H12O6; FW: 180.16) is a very important fuel source to generate universal energy molecules ATP. Glucose level is a key diagnostic parameter for many metabolic disorders. Measurement of glucose can be very important in both research and drug discovery processes. The Glucose Assay Kit provides direct measurement of glucose in various biological samples (e.g., serum, plasma, body fluid, food, growth medium, etc.). Glucose Enzyme Mix specifically oxidizes glucose to generate a product which reacts with a dye to generate color (λ = 570 nm) and fluorescence (Ex/Em = 535/587 nm). The generated color and fluorescence is proportionally to the glucose amount. The method is rapid, simple, sensitive, and suitable for high throughput. The assay is also suitable for monitoring glucose level during fermentation and glucose feeding in protein expression processes. The kit detects 1‐10000μM glucose samples.

II.Component K606-100 Cap Code Part No.

Glucose Assay Buffer 25 ml WM K606-100-1 Glucose Probe (lyophilized) 1 vial Red K606-100-2 Dimethylsulfoxide (DMSO; Dried) 0.4 ml Brown K606-100-3 Glucose Enzyme Mix (lyophilized) 1 vial Green K606-100-4 Glucose Standard (100 nmol/μl) 100 μl Yellow K606-100-5

III. Storage and Handling:

Store kit at –20°C, protect from light. Warm the Glucose Assay Buffer to room temperature and briefly centrifuge vials before opening. Read the entire protocol before performing the assay.

IV. Reagent Preparation:

Glucose Probe: Dissolve in 220 μl DMSO (provided) before use. Store at –20°C, protect from light and moisture. Use within two months. Glucose Enzyme Mix: Dissolve in 220 μl Glucose Assay Buffer. Aliquot and store at – 20°C. Keep on ice while in use. Use within two months.

V. Glucose Assay Protocol:

1. Standard Curve Preparations: For colorimetric assay, dilute the Glucose Standard to 1 nmol/μl by adding 10 μl of the Glucose Standard to 990 μl of Glucose Assay Buffer, mix well. Add 0, 2, 4, 6, 8, 10 μl into each well individually. Adjust volume to 50 μl/well with Glucose Assay Buffer to generate 0, 2, 4, 6, 8, 10 nmol/well of Glucose Standard. For the fluorometric assay, dilute the Glucose Standard solution to 0.1 nmol/μl by adding 10 μl of the Glucose Standard to 990 μl of Glucose Assay Buffer, mix well. Then take 20 μl into 180 μl of Glucose Assay Buffer. Mix well. Add 0, 2, 4, 6, 8, 10 μl into each well individually. Adjust volume to 50 μl/well with Glucose Assay Buffer to generate 0, 0.2, 0.4, 0.6, 0.8, 1.0 nmol/well of the Glucose Standard.
2. Sample Preparations: Prepare test samples in 50 μl/well with Glucose Assay Buffer in a 96‐well plate. If using serum sample, serum (0.5‐2 μl/assay. Normal serum contains ~5 nmol/μl glucose) can be directly diluted in the Glucose Assay Buffer. We suggest testing several doses of your sample to make sure the readings are within the standard curve range.
3. Glucose Reaction Mix: Mix enough reagents for the number of assays to be performed: For each well, prepare a total 50 μl Reaction Mix containing: 46 μl Glucose Assay Buffer 2 μl Glucose Probe* 2 μl Glucose Enzyme Mix

  • Note: The fluorometric assay is 10 times more sensitive than the colorimetric assay. Use

0.4 μl of the probe per reaction will decrease the background reading significantly to increase the detection sensitivity.
4. Mix well. Add 50 μl of the Reaction Mix to each well containing the Glucose Standard and test samples. Mix well.
5. Incubate the reaction for 30 minutes at 37°C, protect from light.
6. Measure O.D. 570nm for colorimetric assay or Ex/Em = 535/590 nm for fluorometric assay in a microplate reader.
7. Calculations: Correct background by subtracting the value derived from the 0 glucose control from all readings (Note: The background reading can be significant and must be subtracted from sample readings). Glucose concentrations of the test samples can then be calculated.
C = Sa/Sv (nmol/μl or μmol/ml, or mM) Where Sa is sample amount (in nmol) from standard curve. Sv is sample volume (in μl) added into the sample wells. Glucose Molecular Weight 180.16.