Team:UT-Tokyo/Protocol

From 2013.igem.org

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<h1 id="Protocols">Protocols</h1>
<h1 id="Protocols">Protocols</h1>
             <h2 id="qRT-PCR">qRT-PCR</h2>
             <h2 id="qRT-PCR">qRT-PCR</h2>
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             <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>
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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>
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3. Dillute the over night culture (1:50) in LB broth containing 100µg/mL ampicillin<br>
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4. grow to OD600=0.6 at 37 degree<br>
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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 08:05, 13 October 2013

           PROTOCOL

Protocols

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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