Team:Warsaw/Calendar-Main/14 May 2008

From 2008.igem.org

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<h4>Michał K.:</h4>
<h4>Michał K.:</h4>
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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>Setup of 9 separate cultures from 9 colonies of transformants pMPMT5+AID (liquid LB+tetracycline).</li>
 
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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> to obtain AID DNA fragment - annealing temperature gradient (from 62&deg;C to 82&deg;C). <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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Template DNA: <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pTrc99A-AID>pTrc99A-AID</a>
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<li> Gel electrophoresis - choice of proper clones (all checked colonies). </li>
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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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Elongation time: 60 sec <br>
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20 cycles </li>
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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). <br>
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Primers:
Primers:
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20 cycles<br>
20 cycles<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 transcription fusion; annealing temperature gradient (from 62&deg;C to 82&deg;C). <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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Template DNA: <i>E. coli</i> <a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#rosetta">Rosetta</a> and <a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#top10">TOP10</a> (negative control) genomic DNA<br>
 
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Elongation time: 4 minutes<br>
 
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20 cycles <br>
 
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<li> Gel electrophoresis of <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#pcr">PCR</a> products(there wasn't any products for T7 RNA polymerase for translation fusion).</li>
 
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<li> Gel electrophoresis of PCR products. </li>
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Revision as of 09:14, 5 October 2008

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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. 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.

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