Team:Warsaw/Calendar-Main/14 May 2008

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<h3>Preparation of <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pMPM-T5-AID>pMPMT5+AID</a> construct</h3>
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<h4>Michał K.</h4>
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<p><ol>
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<li> <a href="https://2008.igem.org/Wiki/Team:Warsaw/protocols#plasmid_DNA_isolation">Isolation</a> of hypothetical <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pMPM-T5-AID>pMPM-T5+AID</a> plasmids from cultures inoculated on previous day. </li>
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<p>Michał K.:</p>
 
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<p>1.Setup of 9 separate cultures from 9 colonies of transformants pMPMT5+AID (liquid LB+tetracycline).<br>
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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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2. 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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<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). <a href="https://2008.igem.org/Team:Warsaw/Calendar-Main/14_May_2008#fig1">Fig. 1</a>.</li></ol>
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Elongation time: 60 sec <br>
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<a name="fig1"><img src="https://static.igem.org/mediawiki/2008/e/e7/Ligation_test_WAW.jpg" width=350/></a>
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<var><b>Fig. 1.</b> 1-DNA ladder; 2 to 9-plasmids isolated from colonies of transformants and digested with HindIII and NcoI</var>
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20 cycles <br>
 
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3. Optimalization 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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<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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<ol>
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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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<a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#T7lLinkB">T7lLinkB</a>
<a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#T7lLinkB">T7lLinkB</a>
<a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#T7pXbSal">T7pXbSal</a>
<a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#T7pXbSal">T7pXbSal</a>
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Template DNA: E. coli Rosetta 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>
Elongation time: 4 minutes
Elongation time: 4 minutes
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20 cycles<br>
20 cycles<br>
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4. Optimalization 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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<li> Gel electrophoresis of PCR products. </li>
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Primers:
 
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</ol></p>
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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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</html>
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Template DNA: E. coli Rosetta and TOP10 (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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5. Gel electrophoresis of PCR products.
 
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</p>
 
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Latest revision as of 13:51, 26 October 2008

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Preparation of pMPMT5+AID construct

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). Fig. 1.
Fig. 1. 1-DNA ladder; 2 to 9-plasmids isolated from colonies of transformants and digested with HindIII and NcoI

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

Michał K.

  1. 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
  2. Gel electrophoresis of PCR products.

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