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Catalog No. M-NTK-301
Microplate Nitrate Test Kit - Standard Range
This nitrate test kit is based on the enzyme
Nitrate Reductase (NaR), catalyzingthe reduction of nitrate to nitrite using the natural electron donor NADH.
The nitrite reacts with color reagents (dyes) under acidic conditions to produce
a visible color. All reaction steps occur in the microplate. The concentration of
nitrate in the unknown sample is determined by measuring absorbance with a
microplate reader using a 540 nm filter (or a filter as close to 540 nm as possible),
and then comparing the absorbance to a standard curve generated from
nitrate standards.
Nitrate can be determined in water samples and extracts of plant tissues, soils
and foods. The test is designed to measure nitrate in the range of 0.5 to 10 ppm
nitrate-N using a 96-well microplate. The nitrate concentration can also be expressed
as μM nitrate, where the range is 36 to 710 μM nitrate.
Nitrite can alsobe determined by omitting NaR and NADH from the test (see
Notes on the Tests,page 4).
IMPORTANT
- Keep contents at 4°C until day of use. See expiration date on box.Supplied in Nitrate Test Kit
•
Assay Buffer in liquid form - (1) 15 ml amber bottle.•
Color Reagent No. 1 in solid form - (1) 15 ml amber bottle.•
Color Reagent No. 2 in solid form - (1) 15 ml amber bottle.•
NADH - 1 tube in amber bag.•
Nitrate Reductase (NaR) - 1 tube in amber bag.•
Nitrate Standard (100 ppm Nitrate-N) in liquid form - 1 tube.•
Quench Agent - 1 tube in amber bag.•
Microplate - (1) 96-well microplate with flat bottom wells.•
Pipetter Reservoir Basins (2) clear plastic containers for reagents.•
Microtubes - 6 tubes for preparing working nitrate standards.Supplied by User
•
25 ml graduated cylinder.•
Pipetters - variable pipetters (10/100 μl and 1 ml) or multi-well pipetter.•
Several test tubes.•
Vortex-type mixer.•
Microplate mixer.•
Microplate reader capable of reading at 540 nm +/- 20 nm.•
Timer (0 to 20 minutes) - a clock or stop watch is adequate.•
Deionized or distilled water (di-water) - Nitrate and Nitrite free!•
2.5 ml of concentrated hydrochloric acid (HCl).•
Ice and Ice Bucket.A. Reagent Preparation
Prepare on day of use. Discard any unused NADH and NaR solutions at end of day.
1
Assay Buffer - ready to use from kit. Warm to room temperature for nitrate tests. Ifdesired, the Assay Buffer may be more quickly warmed in a 30oC water bath.
2 Remove tube of
NaR from amber bag and add 1.0 ml Assay Buffer. Mix for 3 secondswith a vortex-type mixer. Allow to stand at room temperature for 20 minutes, with 3
sec of vortex-mixing at 10 and 20 minutes. Keep on ice until use.
3 Prepare
3 N HCl by adding 2.5 ml concentrated HCl to 7.5 ml di-water. Mix.4 Add
10 ml 3 N HCl to Color Reagent No. 1 bottle. Mix by inverting several times.5 Add
10 ml di-water to Color Reagent No. 2 bottle. Mix by inverting several times.6 Remove tube of
NADH from amber bag and tap tube to settle contents. Add 0.6 ml di-waterand replace cap. Mix for 3 sec with a vortex-type mixer. Keep on ice until use.
7 Add
di-water to the 5 ml line of the clear tube labeled Quench Agent. Mix thoroughly.8 In pipetter reservoir basin (included in kit), add
9 ml Assay Buffer, 1 ml NaR solution(prepared in Step 2), and
0.5 ml NADH solution (prepared in Step 6). Mix thoroughly.B. Preparation of Working Nitrate Standards
Transfer
1 ml of 100 ppm Nitrate-N standard (provided in kit) to a test tube. Dilutewith
9 ml di-water to make a 10 ppm Nitrate-N standard. Use Nitrate standard and the6 microtubes (provided in kit) to prepare nitrate standards as shown below.
Typical Nitrate Std Curve with Slope = 0.085 A-550 nm/ppm Nitrate-N
2
Microplate Nitrate Test Kit (Catalog No. M-NTK-301)Microplate Nitrate Test Kit (Catalog No. M-NTK-301)
35 If using a multi-pipetter, rinse the 2 pipetter reservoir basins with
di-water.Transfer
Color Reagent No. 1 to one basin and Color Reagent No. 2 solutionto the other basin.
6 Add
50 μl Color Reagent No. 1 and 50 μl Color Reagent No. 2 solution toeach well. Shake on a plate mixer for ~10 minutes @ 800 rpm.
7
Zero the plate reader with a Reagent Blank well using a 540 nm filter (or afilter within 20 nm of 540 nm).
8
Read absorbance of all wells. Transfer results to your computer system foranalysis and printing.
D. Calculations
1 To correct for any background absorbance due to the reagents, subtract the
mean absorbance of the reagent blanks from the mean absorbance of each nitrate
standard and unknown sample:
Corrected mean sample A-540 nm = (mean A-540 nm for samples) - (mean A-540 nm for reagent blanks)
2 Generate a standard curve for the nitrate standards (see example at left on
page 2). Using linear graph paper or computer plotting program such as Sigma
Plot® or spreadsheet such as Excel®, plot the ppm nitrate-N on the x-axis, and
the A-540 nm for each nitrate standard on the y-axis. If plotting by hand, draw
a straight line through the points for the nitrate standards. If plotting by computer,
the slope of the line can be calculated for determining nitrate-N ppm in the
unknown sample.
3 Using the standard curve, determine the ppm nitrate-N for the sample: (a)
Find the corrected A-540 nm for the sample on the y-axis of the standard curve.
(b) Follow over along a horizontal line to where the line intersects the standard
curve. Trace down to the x-axis and read the ppm of nitrate-N on the x-axis.
4 The Nitrate Standard Curve shown at the left (on page 2) was prepared using
a plate reader with a 550 nm filter. The slope is 0.085 Abs/ppm Nitrate-N, or
0.019 Abs/ppm Nitrate, or 0.0012 Abs/μM Nitrate.
C. Nitrate Test Procedure
The following procedure uses 2 replicates of each standard, unknown sample
and reagent blank. Using the
Microplate Sample Template, assign and record aset of 2 wells for
each Nitrate Standard, Sample and Reagent Blanks.1 Pipet
10 μl di-water into 2 wells for Reagent Blanks.2 Pipet
10 μl of Working Nitrate Standards and Samples into designatedwells.
3 Pipet
90 μl NaR-Assay Buffer-NADH solution (prepared in Step 8 of ReagentPreparation
) to each well. Shake on a plate mixer for ~20 minutes @ 800rpm.
4 Pipet
30 μl Quench Agent to each well. Shake plate for ~10 min.4
Microplate Nitrate Test Kit (Catalog No. M-NTK-301)NOTES ON THE TEST
Unknown Samples with High Nitrate
This NECi Nitrate Test Kit is capable of determining nitrate levels of up to 10
ppm Nitrate-N (44 ppm Nitrate or 710 μM Nitrate). If an unknown sample is
found to have more than 10 ppm Nitrate-N, the sample may be diluted with diwater1:
10 to allow an exact determination. For example, take 100 μl of sample
and add 900 μl of deionized water to make a 1:10 dilution and then assay 10 μl
of the diluted sample. After finding the amount of nitrate in the diluted sample,
multiply the nitrate concentration by 10 to find the nitrate concentration in the
original sample. NOTE: Keep the sample volume constant by diluting the sample
rather than using a smaller volume of sample in the assay.
Testing-for Nitrite?
Nitrite can be determined by replacing the solution in Step 3 of the
Procedurewith 90 μl of di-water and eliminating Step 4.
Notes on the Reagents
• Assay Buffer - 25 mM K-PO4, 0.025 mM EDTA; pH 7.5
• Color Reagent No. 1 - 1% Sulfanilamide in 3 N HCl
• Color Reagent No. 2 - 0.02% N-Naphthylethylenediamine in di-water
• NADH - ~1.1 mM NADH
• Nitrate Reductase (
NaR) - approx. 0.5 unit of NaR per tube• Nitrate Standard - 1 vial of 100 ppm Nitrate-N
• Quench Agent - mild, non-toxic oxidizing chemical to remove excess NADH.
Caution
Use care when handling
hydrochloric acid (HCl). Gloves are recommended.Waste Disposal
Follow all local guidelines and regulations. If no local guidelines apply to your
situation, wash waste down a sink using large amounts of running water.
