Team:TU Darmstadt/protocols/Colony PCR

From 2013.igem.org

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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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<B> Materials<br></B></font></p>
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<p text-aligne:left style="margin-left:60px; margin-right:50px"><font size="3" color="#F0F8FF" face="Arial regular">
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<br>
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<B>Equipment<br></B>
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<div align="left" style="margin-left:60px; margin-right:50px">
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<ul>
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<li class=list1>- PCR machine</li>
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</ul>
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<B>Chemicals & consumables<br></B>
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<div align="left" style="margin-left:60px; margin-right:50px">
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<li class=list1>- Sterile Eppendorf Tubes</li>
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<li class=list1>- LB-agar plate with appropriate antibiotic</li>
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<li class=list1>- Primers (usually VF2 and VR)</li>
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<li class=list1>- Sterile pipet tips</li>
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</ul>
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</div>
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<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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<B> Procedure<br></B></font></p><br>
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<p text-aligne:left style="margin-left:60px; margin-right:60px"><font size="3" color="#F0F8FF" face="Arial regular">
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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.
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<div align="left" style="margin-left:30px; margin-right:50px">
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<ol>
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<li>Pick one colony with a sterile tip and suspend in 10 µL of ddH2O.</li>
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<li>Inoculate tip with colony into tube. Pipet up and down to ensure all cells are transferred to tube.</li>
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<li>Start the PCR using the following programm and 1X mix.</li>
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<li>Run a gel to determine the product length (don't forget the positiv control).</li>
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</ol>
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</div>
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</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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<B>Mixtures<br></B></font></p><br>
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<B>1X Reaction Mixture<br></B>
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<div align="left" style="margin-left:60px; margin-right:50px">
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<ul>
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<li class=list1>- 2 µL of 10x Thermopol Reaction Buffer</li>
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<li class=list1>- 0,4 µL of dNTPs (10 mM each)</li>
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<li class=list1>- 0,3 µL of Taq DNA Polymerase</li>
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<li class=list1>- VF2 (10 pmol)</li>
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<li class=list1>- 0,3 µL of Taq DNA Polymerase</li>
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<li class=list1>- 0,3 µL of Taq DNA Polymerase</li>
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<li class=list1>- 0,3 µL of Taq DNA Polymerase</li>
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<li class=list1>- 0,3 µL of Taq DNA Polymerase</li>
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</ul>
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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>
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<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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</ol>
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<font size="3" color="#F0F8FF" face="Arial regular">
<font size="3" color="#F0F8FF" face="Arial regular">

Revision as of 19:25, 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).


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)
  • - 0,3 µL of Taq DNA Polymerase
  • - 0,3 µL of Taq DNA Polymerase
  • - 0,3 µL of Taq DNA Polymerase
  • - 0,3 µL of Taq DNA Polymerase


References

  1. Mandel, M. and Higa, A.: Calcium-dependent bacteriophage DNA infection. J Mol Biol, 1970, 53, 159-162

Short explanation
 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.

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

Procedure
 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)

Reaction Mix
 1X reaction mix contains:
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:

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