Difference between revisions of "Team:TUST China/Attributions"

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            <h1 style="top: 160px;text-align: center">Detection of Tetracycline</h1>
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            <hr style="height: 2px;background-color: black; width: 70%">
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        </div>
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        <div class="container content-cen">
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            <h2>Sample Pretreatment</h2>
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            <p >
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                Aquaculture, fish pond water samples and stool samples. (From the area with high tetracycline content. Water samples need to be protected from light, and the following operations are required to avoid light.)
 +
            </p>
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            <h2>Pretreatment of Water Samples:</h2>
 +
            <p>1. Centrifuge the water sample in the 50ml centrifuge tube（8000-10000r/min, 5min）and take the supernatant in the clean centrifuge tube. Repeat many times until there is no visible impurity. (Repeat this procedure no less than three times.)</p>
 +
            <p>2. The water sample in the centrifugal tube is passed through the membrane and packed into 10 clean centrifugal tubes, each containing 10 ml.</p>
 +
            <p>3. Placing 10 centrifuge tubes at -80 degrees Celsius for no less than 24 hours.</p>
 +
            <p>4. The samples were sealed with preservative film after 24 hours of freezing, and several small holes were punctured. The samples were lyophilized by freeze-drying machine until they were all turned into powder.</p>
 +
            <h2>Pretreatment of Feces</h2>
 +
            <p>1. Take appropriate amount of fecal samples in 50 ml centrifugal tube with water solution, centrifugal(8000-10000r/min，5min）, take supernatant in a clean centrifugal tube. Adding water to dissolve, centrifugation
 +
                （8000-10000r/min，5min）, and take the supernatant in centrifugation. (Repeated operation above, all the supernatant was placed in centrifuge
 +
                tube.
 +
            </p>
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            <p>2. The supernatant will be centrifugally repeated several times until there is no visible impurity.</p>
 +
            <p>3. The samples in the centrifugal tube were passed through the mmbrane and packed into 10 clean centrifugal tubes, each containing 10 ml.</p>
 +
            <p>4. Placing 10 centrifuge tubes at -80 degrees Celsius for less than 24 hours.</p>
 +
            <p>5. Freezing the sample after 24 hours with a plastic wrap and seal a plurality of small holes.</p>
 +
            <p>6. The lyophilized machine was used for freeze-drying operations until all the samples were changed into powder.</p>
 +
            <h2>Subsequent Operations After Freeze Drying</h2>
 +
            <p>1. The freeze-dried powder is merged into a centrifugal tube, dissolved in a small amount of water (just dissolved, water cannot be excessive), and put in - 80 degrees Celsius freezing 24 hours, freeze-dried.</p>
 +
            <p>2. Adding 1mlDMF to dissolve the powder (over film treatment) and get the final samples. (Need to operate in hood)</p>
 +
            <h2>Detection: High Performance Liquid Chromatography</h2>
 +
            <p>1. Instrument: WatersC18ODS-3 column (250 mm 4.6 mm, 5 m).</p>
 +
            <p>2. Reagents:</p>
 +
            <p>Mobile phase A solution: methanol (filtration treatment) B liquid: ddH2O</p>
 +
            <p>Note:</p>
 +
            <p>Ultrasound is needed for at least half an hour before use and the temperature should not be too high.
 +
                Injection needles need to be cleaned by mobile phase. Use brown liquid vial
 +
            </p>
 +
            <p>3. Setting speed: 1 mL/minColumn <p>temperature: 35 degrees Celsius</p> <p>Absorption peak: 355nm</p>
 +
 +
            <p>Injection volume: 20ul</p>
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            </p>
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            <p>Setting Concentration Gradient：</p>
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            <table class="table table-striped" >
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                <thead>
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                    <tr>
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                        <td>Time</td>
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                        <td>A</td>
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                        <td>B</td>
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                    </tr>
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                </thead>
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                <tbody>
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                <tr>
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                    <td style="border-right: solid 2px #add9c0;">0-2min</td>
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                    <td>5%</td>
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                    <td>95%</td>
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                </tr>
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                <tr>
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                    <td style="border-right: solid 2px #add9c0;">2.1min</td>
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                    <td>40%</td>
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                    <td>60%</td>
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                </tr>
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                <tr>
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                    <td style="border-right: solid 2px #add9c0;">8min</td>
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                    <td>70%</td>
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                    <td>30%</td>
 +
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                </tr>
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                <tr>
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                    <td>8.1-12min</td>
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                    <td>90%</td>
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                    <td>10%</td>
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 +
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                </tr>
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                <tr>
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                    <td>12-15min</td>
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                    <td>5％</td>
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                    <td>95%</td>
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 +
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                </tr>
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                </tbody>
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            </table>
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            <p>This method should peak at about 6.72min.</p>
 +
            <p class="lead">Please keep all the data and images of the liquid phase and send the results to TUST_China</p>
 +
            <p><i class="footiconfont bigicon">&#xe609;&nbsp;&nbsp;tustigem2018@163.com</i></p>
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Revision as of 15:30, 12 October 2018

nav

achievement attibution

Detection of Tetracycline

Sample Pretreatment

Aquaculture, fish pond water samples and stool samples. (From the area with high tetracycline content. Water samples need to be protected from light, and the following operations are required to avoid light.)

Pretreatment of Water Samples:

1. Centrifuge the water sample in the 50ml centrifuge tube（8000-10000r/min, 5min）and take the supernatant in the clean centrifuge tube. Repeat many times until there is no visible impurity. (Repeat this procedure no less than three times.)

2. The water sample in the centrifugal tube is passed through the membrane and packed into 10 clean centrifugal tubes, each containing 10 ml.

3. Placing 10 centrifuge tubes at -80 degrees Celsius for no less than 24 hours.

4. The samples were sealed with preservative film after 24 hours of freezing, and several small holes were punctured. The samples were lyophilized by freeze-drying machine until they were all turned into powder.

Pretreatment of Feces

1. Take appropriate amount of fecal samples in 50 ml centrifugal tube with water solution, centrifugal(8000-10000r/min，5min）, take supernatant in a clean centrifugal tube. Adding water to dissolve, centrifugation （8000-10000r/min，5min）, and take the supernatant in centrifugation. (Repeated operation above, all the supernatant was placed in centrifuge tube.

2. The supernatant will be centrifugally repeated several times until there is no visible impurity.

3. The samples in the centrifugal tube were passed through the mmbrane and packed into 10 clean centrifugal tubes, each containing 10 ml.

4. Placing 10 centrifuge tubes at -80 degrees Celsius for less than 24 hours.

5. Freezing the sample after 24 hours with a plastic wrap and seal a plurality of small holes.

6. The lyophilized machine was used for freeze-drying operations until all the samples were changed into powder.

Subsequent Operations After Freeze Drying

1. The freeze-dried powder is merged into a centrifugal tube, dissolved in a small amount of water (just dissolved, water cannot be excessive), and put in - 80 degrees Celsius freezing 24 hours, freeze-dried.

2. Adding 1mlDMF to dissolve the powder (over film treatment) and get the final samples. (Need to operate in hood)

Detection: High Performance Liquid Chromatography

1. Instrument: WatersC18ODS-3 column (250 mm 4.6 mm, 5 m).

2. Reagents:

Mobile phase A solution: methanol (filtration treatment) B liquid: ddH2O

Note:

Ultrasound is needed for at least half an hour before use and the temperature should not be too high. Injection needles need to be cleaned by mobile phase. Use brown liquid vial

3. Setting speed: 1 mL/minColumn

temperature: 35 degrees Celsius

Absorption peak: 355nm

Injection volume: 20ul

Setting Concentration Gradient：

Time A B
0-2min 5% 95%
2.1min 40% 60%
8min 70% 30%
8.1-12min 90% 10%
12-15min 5％ 95%

This method should peak at about 6.72min.

Please keep all the data and images of the liquid phase and send the results to TUST_China

  tustigem2018@163.com