Team:TU Darmstadt/protocols/Colony PCR

From 2013.igem.org

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<div align="left" style="margin-left:30px; margin-right:50px">
<ol>
<ol>
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<li>Pick one colony with a sterile tip and suspend in 10 µL of ddH2O.</li>
+
<li>Pick one colony with a sterile tip and suspend in 10 µL of ddH<sub>2</sub>O.</li>
<li>Inoculate tip with colony into tube. Pipet up and down to ensure all cells are transferred to tube.</li>
<li>Inoculate tip with colony into tube. Pipet up and down to ensure all cells are transferred to tube.</li>
<li>Start the PCR using the following programm and 1X mix.</li>
<li>Start the PCR using the following programm and 1X mix.</li>
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</font></p><br>
</font></p><br>
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<p text-aligne:left style="margin-left:50px; margin-right:50px"><font size="4.5" color="#F0F8FF" face="Arial regular">
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<table border="0">
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  <tr>
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    <th>#</th>
 +
    <th>Temperature</th>
 +
    <th>Time</th>
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  </tr>
 +
  <tr>
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    <td>1</td>
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    <td>95 °C</td>
 +
    <td>00:05:00</td>
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  </tr>
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  <tr>
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    <td>2</td>
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    <td>95 °C</td>
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    <td>00:00:20</td>
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  </tr>
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  <tr>
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    <td>3</td>
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    <td>62 °C</td>
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    <td>00:00:30</td>
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  </tr>
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  <tr>
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    <td>4</td>
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    <td>68 °C</td>
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    <td>00:02:00</td>
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  </tr>
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  <tr>
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    <td>5</td>
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    <td>GO TO 2</td>
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    <td>REPEAT 30x</td>
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  </tr>
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  <tr>
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    <td>6</td>
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    <td>68 °C</td>
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    <td>00:05:00</td>
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  </tr>
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  <tr>
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    <td>7</td>
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    <td>4 °C</td>
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    <td>HOLD</td>
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  </tr>
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</table>
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<li class=list1>- 0,3 µL of Taq DNA Polymerase</li>
<li class=list1>- 0,3 µL of Taq DNA Polymerase</li>
<li class=list1>- VF2 (10 pmol)</li>
<li class=list1>- VF2 (10 pmol)</li>
-
<li class=list1>- 0,3 µL of Taq DNA Polymerase</li>
+
<li class=list1>- VR (10 pmol) </li>
-
<li class=list1>- 0,3 µL of Taq DNA Polymerase</li>
+
<li class=list1>- 0,6 µL of DMSO </li>
-
<li class=list1>- 0,3 µL of Taq DNA Polymerase</li>
+
<li class=list1>- 1 µL of colony suspension </li>
-
<li class=list1>- 0,3 µL of Taq DNA Polymerase</li>
+
<li class=list1>- ddH<sub>2</sub>O to 20 µL </li>
</ul>
</ul>

Revision as of 19:50, 4 October 2013





Colony PCR

Materials


Equipment

  • - PCR machine


Chemicals & consumables

  • - Sterile Eppendorf Tubes
  • - LB-agar plate with appropriate antibiotic
  • - Primers (usually VF2 and VR)
  • - Sterile pipet tips


Procedure


The colony PCR is a modified PCR programm employed to verifiy transformation success by amplifiying the insert or the vector construct used for transformation. This is necessary due to the fact, that a transformation with the empty vector may lead to antibiotic resistance.

  1. Pick one colony with a sterile tip and suspend in 10 µL of ddH2O.
  2. Inoculate tip with colony into tube. Pipet up and down to ensure all cells are transferred to tube.
  3. Start the PCR using the following programm and 1X mix.
  4. Run a gel to determine the product length (don't forget the positiv control).


# Temperature Time
1 95 °C 00:05:00
2 95 °C 00:00:20
3 62 °C 00:00:30
4 68 °C 00:02:00
5 GO TO 2 REPEAT 30x
6 68 °C 00:05:00
7 4 °C HOLD

Mixtures


1X Reaction Mixture

  • - 2 µL of 10x Thermopol Reaction Buffer
  • - 0,4 µL of dNTPs (10 mM each)
  • - 0,3 µL of Taq DNA Polymerase
  • - VF2 (10 pmol)
  • - VR (10 pmol)
  • - 0,6 µL of DMSO
  • - 1 µL of colony suspension
  • - ddH2O to 20 µL


PCR programm:

Retrieved from "http://2013.igem.org/Team:TU_Darmstadt/protocols/Colony_PCR"