Team:Warsaw/Calendar-Main/14 May 2008

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<h3>Preparation of pMPMT5+AID construct and PCRs for fusions</h3>
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<h3>Preparation of pMPMT5+AID construct and PCRs for fusions<br>
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<h4>Michał K.:</h4>
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Michał K.</h3>
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<p><ol>
<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>
<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> Gel electrophoresis - choice of proper clones (all checked colonies). </li>
<li> Gel electrophoresis - choice of proper clones (all checked colonies). </li>
<img src="https://static.igem.org/mediawiki/2008/e/e7/Ligation_test_WAW.jpg" width=350/>
<img src="https://static.igem.org/mediawiki/2008/e/e7/Ligation_test_WAW.jpg" width=350/>
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<h3>1-DNA ladder; 2 to 9-plasmids isolated from 8 colonies of transformants and digested with HindIII and NcoI</h3>
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<var>1-DNA ladder; 2 to 9-plasmids isolated from 8 colonies of transformants and digested with HindIII and NcoI</var>
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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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<li> Optimization of conditions for <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>
Primers:
Primers:
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<li> Gel electrophoresis of PCR products. </li>
<li> Gel electrophoresis of PCR products. </li>
<img src="https://static.igem.org/mediawiki/2008/6/6c/T7grad_WAW2.jpg" width=350/>
<img src="https://static.igem.org/mediawiki/2008/6/6c/T7grad_WAW2.jpg" width=350/>
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<h3>PCR products. 2-annealing temperature 62&deg;C, 6-annealing temperature 82&deg;C</h3>
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<var>PCR products. 2-annealing temperature 62&deg;C, 6-annealing temperature 82&deg;C</var>
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Revision as of 13:59, 9 October 2008

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Preparation of pMPMT5+AID construct and PCRs for fusions
Michał K.

  1. Isolation of hypothetical pMPM-T5+AID plasmids from cultures inoculated on previous day.
  2. Restriction digest of plasmids with HindIII and NcoI (1xTango buffer) - construct control.
  3. Gel electrophoresis - choice of proper clones (all checked colonies).
    4. 1-DNA ladder; 2 to 9-plasmids isolated from 8 colonies of transformants and digested with HindIII and NcoI
  4. Optimization of conditions for PCR - T7 RNA polymerase for translation fusion; annealing temperature gradient (from 62°C to 82°C); Taq polymerase with supplemented buffer (Łukasz Banasiak helped us with that problematic PCR).
    Primers: T7lLinkB T7pXbSal
    Template DNA: E. coli Rosetta genomic DNA
    Elongation time: 4 minutes
    20 cycles
  5. Gel electrophoresis of PCR products.
    6. PCR products. 2-annealing temperature 62°C, 6-annealing temperature 82°C

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