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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>
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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>
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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.<br>
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| - | <br>
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| - | <B> Materials<br></B>
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| - | Sterile Eppendorf Tubes<br>
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| - | LB-agar plate with appropriate antibiotic<br>
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| - | Primers (usually VF2 and VR)<br>
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| - | PCR machine<br>
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| - | Sterile pipet tips<br>
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| - | <br>
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| - | <B> Procedure<br></B>
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| - | 1. Pick one colony with a sterile tip and suspend in 10 µL of ddH2O<br>
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| - | 2. Inoculate tip with colony into tube. Pipet up and down to ensure all cells are transferred to tube.<br>
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| - | 3. Start the PCR using the following programm and 1X mix<br>
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| - | 4. Run a gel to determine the product length (don't forget the positiv control)<br>
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| - | <br>
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| - | <B> Reaction Mix<br></B>
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| - | 1X reaction mix contains:<br>
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| - | 2 µL of 10x Thermopol Reaction Buffer<br>
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| - | 0,4 µL of dNTPs (10 mM each)<br>
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| - | 0,3 µL of Taq DNA Polymerase<br>
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| - | VF2 (10 pmol)<br>
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| - | VR (10 pmol)<br>
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| - | 0,6 µL of DMSO<br>
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| - | 1 µL of colony suspension<br>
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| - | ddH2O to 20 µL<br>
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| | PCR programm: | | PCR programm: |
Colony PCR
Materials
Equipment
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.
- Pick one colony with a sterile tip and suspend in 10 µL of ddH2O.
- Inoculate tip with colony into tube. Pipet up and down to ensure all cells are transferred to tube.
- Start the PCR using the following programm and 1X mix.
- 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
PCR programm:
"
