Team:TU Darmstadt/protocols/Colony PCR

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<img alt="Problem" src="/wiki/images/6/66/Darmstadt_green_Problem.jpg" width="100" height="30"></a>
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<font size="8" color="#F0F8FF" face="Arial regular">Materials |</font>
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<font size="8" color="#F0F8FF" face="Arial regular">Protocols</font>
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<!-- Colony PCR -->
<!-- Colony PCR -->
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<br>
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<h2><font size="6" color="#F0F8FF" face="Arial regular"> Colony PCR </font></h2>
<h2><font size="6" color="#F0F8FF" face="Arial regular"> Colony PCR </font></h2>
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<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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  <tr>
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    <th><font size="3" color="#F0F8FF" face="Arial regular">#</font></th>
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    <th><font size="3" color="#F0F8FF" face="Arial regular">Temperature</font></th>
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    <th><font size="3" color="#F0F8FF" face="Arial regular">Time</font></th>
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    <td><font size="3" color="#F0F8FF" face="Arial regular">1</font></td>
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    <td><font size="3" color="#F0F8FF" face="Arial regular">95 °C</font></td>
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    <td><font size="3" color="#F0F8FF" face="Arial regular">00:05:00</font></td>
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  </tr>
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  <tr>
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    <td><font size="3" color="#F0F8FF" face="Arial regular">2</font></td>
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    <td><font size="3" color="#F0F8FF" face="Arial regular">95 °C</font></td>
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    <td><font size="3" color="#F0F8FF" face="Arial regular">00:00:20</font></td>
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  </tr>
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  <tr>
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    <td><font size="3" color="#F0F8FF" face="Arial regular">3</font></td>
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    <td><font size="3" color="#F0F8FF" face="Arial regular">62 °C</font></td>
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    <td><font size="3" color="#F0F8FF" face="Arial regular">00:00:30</font></td>
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  </tr>
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  <tr>
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    <td><font size="3" color="#F0F8FF" face="Arial regular">4</font></td>
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    <td><font size="3" color="#F0F8FF" face="Arial regular">68 °C</font></td>
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    <td><font size="3" color="#F0F8FF" face="Arial regular">00:02:00</font></td>
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  </tr>
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  <tr>
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    <td><font size="3" color="#F0F8FF" face="Arial regular">5</font></td>
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    <td><font size="3" color="#F0F8FF" face="Arial regular">GO TO 2</font></td>
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    <td><font size="3" color="#F0F8FF" face="Arial regular">REPEAT 30x</font></td>
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    <td><font size="3" color="#F0F8FF" face="Arial regular">6</font></td>
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    <td><font size="3" color="#F0F8FF" face="Arial regular">68 °C</font></td>
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    <td><font size="3" color="#F0F8FF" face="Arial regular">00:05:00</font></td>
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  </tr>
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  <tr>
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    <td><font size="3" color="#F0F8FF" face="Arial regular">7</font></td>
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    <td><font size="3" color="#F0F8FF" face="Arial regular">4 °C</font></td>
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    <td><font size="3" color="#F0F8FF" face="Arial regular">HOLD</font></td>
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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>
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<li class=list1>- VR (10 pmol) </li>
-
<li class=list1>- 0,3 µL of Taq DNA Polymerase</li>
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<li class=list1>- 0,6 µL of DMSO </li>
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<li class=list1>- 0,3 µL of Taq DNA Polymerase</li>
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<li class=list1>- 1 µL of colony suspension </li>
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<li class=list1>- 0,3 µL of Taq DNA Polymerase</li>
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<li class=list1>- ddH<sub>2</sub>O to 20 µL </li>
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<h2><font size="6" color="#F0F8FF" face="Arial regular">References</font></h2>
 
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<font size="3" color="#F0F8FF" face="Arial regular">
 
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<ol>
 
-
<li style="margin-left:15px; margin-right:50px; text-align:justify">Mandel, M. and Higa, A.: <i>Calcium-dependent bacteriophage DNA infection</i>. J Mol Biol, 1970, 53, 159-162</li>
 
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</font>
 
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<font size="3" color="#F0F8FF" face="Arial regular">
 
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<p text-aligne:left style="margin-left:50px; margin-right:50px">
 
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<B> Short explanation<br></B>
 
-
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.<br>
 
-
<br>
 
-
<B> Materials<br></B>
 
-
Sterile Eppendorf Tubes<br>
 
-
LB-agar plate with appropriate antibiotic<br>
 
-
Primers (usually VF2 and VR)<br>
 
-
PCR machine<br>
 
-
Sterile pipet tips<br>
 
-
<br>
 
-
<B> Procedure<br></B>
 
-
1. Pick one colony with a sterile tip and suspend in 10 µL of ddH2O<br>
 
-
2. Inoculate tip with colony into tube. Pipet up and down to ensure all cells are transferred to tube.<br>
 
-
3. Start the PCR using the following programm and 1X mix<br>
 
-
4. Run a gel to determine the product length (don't forget the positiv control)<br>
 
-
<br>
 
-
 
-
<B> Reaction Mix<br></B>
 
-
1X reaction mix contains:<br>
 
-
2 µL of 10x Thermopol Reaction Buffer<br>
 
-
0,4 µL of dNTPs (10 mM each)<br>
 
-
0,3 µL of Taq DNA Polymerase<br>
 
-
VF2 (10 pmol)<br>
 
-
VR (10 pmol)<br>
 
-
0,6 µL of DMSO<br>
 
-
1 µL of colony suspension<br>
 
-
ddH2O to 20 µL<br>
 
-
 
-
PCR programm:
 

Latest revision as of 00:31, 5 October 2013







Lab book | Materials | Protocols

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


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