Team:Warsaw/Calendar-Main/15 May 2008

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<html><h3>Preparation of <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pMPM-T5%2BAID%2BT7>pMPMT5-AID+T7</a> and <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pMPM-T5%2BAIDT7>pMPMT5+AID-T7</a></h3>
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<h4>Michał K.</h4>
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<h4>Michał K.:</h4>
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<p><ol>
<p><ol>
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<li> <a href="https://2008.igem.org/Wiki/Team:Warsaw/protocols#plasmid_DNA_isolation">Isolation</a> of hypothetical pMPM-T5+AID plasmids from cultures inoculated on previous day.</li>
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<li> <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#pcr">PCR</a> - AID for translational fusion. <br>
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Primers:
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<li> Restriction <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#digest">digest</a> of plasmids with HindIII and NcoI (1xTango buffer) - construct control.</li>
 
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<a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#AIDlNrH">AIDlNrH</a>
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<a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#AIDpLinB">AIDpLinB</a>
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<br>
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Template DNA: <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pTrc99A-AID>pTrc99A-AID</a>
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<br>
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Annealing temperature: 55&deg;C<br>
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Elongation time: 60 s<br>
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20 cycles
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</li>
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<li> Gel electrophoresis - choice of proper clones (all checked colonies). </li>
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<li> <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#pcr">PCR</a> - T7 RNA-polymerase for translational fusion; Taq polymerase with supplemented buffer (Łukasz Banasiak helped us with that problematic PCR).<br>
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<li> Optimization of conditions for <html><a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#pcr">PCR</a> - T7 RNA polymerase for translation fusion; annealing temperature gradient (from 62&deg;C to 82&deg;C); Taq polymerase with supplemented buffer (Łukasz Banasiak helped us with that problematic PCR). <br>
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Primers:
Primers:
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Template DNA: <i>E. coli</i> <a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#rosetta">Rosetta</a> genomic DNA<br>
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Template DNA: <i>E. coli</i> <a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#rosetta">Rosetta</a> genomic DNA<br>
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Elongation time: 4 minutes
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Annealing temperature: 62&deg;C<br>
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<br>
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Elongation time: 4 minutes<br>
20 cycles<br>
20 cycles<br>
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</li>
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<li> <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#pcr">PCR</a> - T7 RNA-polymerase for transcriptional fusion.<br>
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Primers:
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<a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#T7lRBSHi">T7lRBSHi</a>
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<a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#T7pXbSal">T7pXbSal</a>
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<br>
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Template DNA: <i>E. coli</i> <a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#rosetta">Rosetta</a>  genomic DNA<br>
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Annealing temperature: 62&deg;C<br>
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Elongation time: 4 minutes<br>
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<li> Gel electrophoresis of PCR products. </li>
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20 cycles
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</ol></p>
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</li>
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</p>
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Latest revision as of 13:46, 26 October 2008

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Preparation of pMPMT5-AID+T7 and pMPMT5+AID-T7

Michał K.

  1. PCR - AID for translational fusion.
    Primers: AIDlNrH AIDpLinB
    Template DNA: pTrc99A-AID
    Annealing temperature: 55°C
    Elongation time: 60 s
    20 cycles
  2. PCR - T7 RNA-polymerase for translational fusion; Taq polymerase with supplemented buffer (Łukasz Banasiak helped us with that problematic PCR).
    Primers: T7lLinkB T7pXbSal
    Template DNA: E. coli Rosetta genomic DNA
    Annealing temperature: 62°C
    Elongation time: 4 minutes
    20 cycles
  3. PCR - T7 RNA-polymerase for transcriptional fusion.
    Primers: T7lRBSHi T7pXbSal
    Template DNA: E. coli Rosetta genomic DNA
    Annealing temperature: 62°C
    Elongation time: 4 minutes
    20 cycles

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