Team:UT-Tokyo/Protocol

From 2013.igem.org

(Difference between revisions)
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                     <li><span class="mhead">Protocols</span>
                     <li><span class="mhead">Protocols</span>
                     <ol class="menu">
                     <ol class="menu">
 +
                        <li><a href="#Transformation">Transformation</a></li>
 +
                        <li><a href="#Electrophoresis">Electrophoresis</a></li>
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                        <li><a href="#Colony PCR">Colony PCR</a></li>
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                        <li><a href="#Miniprep">Miniprep</a></li>
 +
                        <li><a href="#">Digestion</a>Digestion</li>
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                        <li><a href="#">Gel extraction</a>Gel extraction</li>
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                        <li><a href="#">Ligation</a>Ligation</li>
                         <li><a href="#qRT-PCR">qRT-PCR</a></li>
                         <li><a href="#qRT-PCR">qRT-PCR</a></li>
                     </ol>
                     </ol>
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</p>
</p>
<h1 id="Protocols">Protocols</h1>
<h1 id="Protocols">Protocols</h1>
 +
 +
            <h2 id="Transformation">Transformation</h2>
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            <p class="ini"> 1. Put the competent cell on ice and leave it until disolution.
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2. Add DNA 1µL to competent cell 25µL on 2ml tube.
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3. Place on ice for 20min.
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4. Heatshock at 42℃ for 45sec.
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5. Place the tube on ice for 3 minutes.
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6. Add 100µL of LB medium and place tube on 37°C for 20min
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7. Plate 100µL of the medium and spread well.
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8. Incubate the plate on 37℃. </p>
 +
 +
            <h2 id="Electrophoresis">Electrophoresis</h2>
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            <p class="ini">  </p>
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            <h2 id="Colony PCR">Colony PCR</h2>
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            <p class="ini">  </p>
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 +
            <h2 id="Miniprep">Miniprep</h2>
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            <p class="ini">  </p>
 +
 +
            <h2 id="Digestion">Digestion</h2>
 +
            <p class="ini">  </p>
 +
 +
            <h2 id="Gel extraction">Gel extraction</h2>
 +
            <p class="ini">  </p>
 +
 +
            <h2 id="Ligation">Ligation</h2>
 +
            <p class="ini">  </p>
 +
             <h2 id="qRT-PCR">qRT-PCR</h2>
             <h2 id="qRT-PCR">qRT-PCR</h2>
-
             <p class="ini"> 1. Pick up a colony and inoculate in LB broth containing 100µg/mL ampicillin<br>
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             <p class="ini"> 1. Pick up a colony and inoculate in LB broth containing 100µg/mL ampicillin.<br>
-
2. Incubate at 37 degree over night<br>
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2. Incubate at 37 degree over night.<br>
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3. Dillute the over night culture (1:50) in LB broth containing 100µg/mL ampicillin<br>
+
3. Dillute the over night culture (1:50) in LB broth containing 100µg/mL ampicillin.<br>
-
4. Grow to OD600=0.6 at 37 degree<br>
+
4. Grow to OD600=0.6 at 37 degree.<br>
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5. Centrifuge 1mL culture at 15,000rpm for 1min<br>
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5. Centrifuge 1mL culture at 15,000rpm for 1min.<br>
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6. Remove the supernatant<br>
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6. Remove the supernatant.<br>
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7. Resuspend with TE buffer containing lysozyme(0.4mg/mL)<br>
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7. Resuspend with TE buffer containing lysozyme(0.4mg/mL).<br>
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8. Incubate at room temperature for 5min<br>
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8. Incubate at room temperature for 5min.<br>
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9. Isolate total RNA from the lysate with RNeasy® Mini<br>
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9. Isolate total RNA from the lysate with RNeasy® Mini.<br>
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10. Synthesize cDNA from 1µg total RNA with PrimeScript® 1st strand cDNA Synthesis Kit<br>
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10. Synthesize cDNA from 1µg total RNA with PrimeScript® 1st strand cDNA Synthesis Kit.<br>
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11. Quantify the cDNA of the target gene or 16S rRNA with Power SYBR® Green PCR Master Mix </p>
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11. Quantify the cDNA of the target gene or 16S rRNA with Power SYBR® Green PCR Master Mix. </p>
         </div>
         </div>

Revision as of 08:33, 13 October 2013

           PROTOCOL

Protocols

Transformation

1. Put the competent cell on ice and leave it until disolution. 2. Add DNA 1µL to competent cell 25µL on 2ml tube. 3. Place on ice for 20min. 4. Heatshock at 42℃ for 45sec. 5. Place the tube on ice for 3 minutes. 6. Add 100µL of LB medium and place tube on 37°C for 20min 7. Plate 100µL of the medium and spread well. 8. Incubate the plate on 37℃.

Electrophoresis

Colony PCR

Miniprep

Digestion

Gel extraction

Ligation

qRT-PCR

1. Pick up a colony and inoculate in LB broth containing 100µg/mL ampicillin.
2. Incubate at 37 degree over night.
3. Dillute the over night culture (1:50) in LB broth containing 100µg/mL ampicillin.
4. Grow to OD600=0.6 at 37 degree.
5. Centrifuge 1mL culture at 15,000rpm for 1min.
6. Remove the supernatant.
7. Resuspend with TE buffer containing lysozyme(0.4mg/mL).
8. Incubate at room temperature for 5min.
9. Isolate total RNA from the lysate with RNeasy® Mini.
10. Synthesize cDNA from 1µg total RNA with PrimeScript® 1st strand cDNA Synthesis Kit.
11. Quantify the cDNA of the target gene or 16S rRNA with Power SYBR® Green PCR Master Mix.

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