Team:Warsaw/Calendar-Main/13 May 2008

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<html><h3>Preparation of pMPMT5+AID construct and PCRs for fusions<br>
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<html><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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Michał K.</h3>
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<h4>Michał K.</h4>
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Setup of separate cultures from colonies of transformants <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pMPM-T5-AID>pMPMT5+AID</a> (liquid LB+tetracycline).</p>
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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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<li>Setup of 8 separate cultures from 8 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). <a href="https://2008.igem.org/Team:Warsaw/Calendar-Main/13_May_2008#fig1">Fig. 1</a>.<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> to obtain AID DNA fragment - annealing temperature gradient (from 62&deg;C to 82&deg;C). <br>
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Primers:
Primers:
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20 cycles </li>
20 cycles </li>
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<img src="https://static.igem.org/mediawiki/2008/7/71/Aid_pcr_grad_WAW.jpg" width=350/><var>1-DNA ladder; 2 to 13-PCR products (2-annealing temperature 62&deg;C, 13-annealing temperature 82&deg;C)</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). <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 translational fusion; annealing temperature gradient (from 62&deg;C to 82&deg;C). <a href="https://2008.igem.org/Team:Warsaw/Calendar-Main/13_May_2008#fig2">Fig. 2</a>.<br>
Primers:
Primers:
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20 cycles<br>
20 cycles<br>
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</li>
</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 transcription fusion; annealing temperature gradient (from 62&deg;C to 82&deg;C). <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 transcriptional fusion; annealing temperature gradient (from 62&deg;C to 82&deg;C). <a href="https://2008.igem.org/Team:Warsaw/Calendar-Main/13_May_2008#fig2">Fig. 2</a>. <br>
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> 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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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 - <a href="https://2008.igem.org/Team:Warsaw/Calendar-Main/13_May_2008#fig3">Fig. 3</a>.) genomic DNA<br>
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20 cycles <br>
20 cycles <br>
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<li> Gel electrophoresis of PCR products (there wasn't any products for T7 RNA polymerase for translation fusion).</li>
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<li> Gel electrophoresis of PCR products (there weren't any products for T7 RNA polymerase for translational fusion).</li>
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<img src="https://static.igem.org/mediawiki/2008/9/97/T7grad_WAW1.jpg" width=350/>
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</p>
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<var>PCR products: upper - for transcription fusion, lower - for translation fusion; 1-DNA ladder; 2-annealing temperature 62&deg;C; 12-annealing temperature 82&deg;C.</var>
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<img src="https://static.igem.org/mediawiki/2008/6/6c/T7grad_WAW2.jpg" width=350/>
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<a name="fig1"><img src="https://static.igem.org/mediawiki/2008/7/71/Aid_pcr_grad_WAW.jpg" width=350/></a><var><b>Fig. 1.</b> Gradient PCR products for AID:<br> <b>1</b> -DNA ladder; <b>2 to 13</b> -PCR products: In lane <b>2</b> is product of PCR reaction with annealing temperature 62&deg;C (the lowest temperature of gradient), in lane <b>13</b> is product of PCR reaction with annealing temperature 82&deg;C (the highest temperature of gradient). The lanes between them contain products of PCR with middle annealing temperatures.</var>
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<var>PCR products - negative control (Top10 genomic DNA). 2-annealing temperature 62&deg;C, 6-annealing temperature 82&deg;C</var>
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<a name="fig2"><img src="https://static.igem.org/mediawiki/2008/9/97/T7grad_WAW1.jpg" width=350/></a>
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<var><b>Fig. 2.</b> Gradient PCR products: upper - for transcriptional fusion, lower - for translational fusion; 1-DNA ladder; 2 to 12-PCR products-border annealing temperatures of gradient: 62&deg;C (lane 2) to 82&deg;C (lane 12)</var>
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<a name="fig3"><img src="https://static.igem.org/mediawiki/2008/6/6c/T7grad_WAW2.jpg" width=350/></a>
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<var><b>Fig. 3.</b> Gradient PCR products for  negative control (Top10 genomic DNA): 1-DNA ladder; 2 to 6-PCR products-border annealing temperatures of gradient: 62&deg;C (lane 2) to 82&deg;C (lane 6).</var>
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Preparation of pMPMT5+AID construct

Michał K.

Setup of separate cultures from colonies of transformants pMPMT5+AID (liquid LB+tetracycline).

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

Michał K.

  1. Optimization of conditions for PCR to obtain AID DNA fragment - annealing temperature gradient (from 62°C to 82°C). Fig. 1.
    Primers: AIDlNrH AIDpLinB
    Template DNA: pTrc99A-AID
    Elongation time: 60 sec
    20 cycles
  2. Optimization of conditions for PCR - T7 RNA polymerase for translational fusion; annealing temperature gradient (from 62°C to 82°C). Fig. 2.
    Primers: T7lLinkB T7pXbSal
    Template DNA: E. coli Rosetta genomic DNA
    Elongation time: 4 minutes
    20 cycles
  3. Optimization of conditions for PCR - T7 RNA polymerase for transcriptional fusion; annealing temperature gradient (from 62°C to 82°C). Fig. 2.
    Primers: T7lRBSHi T7pXbSal
    Template DNA: E. coli Rosetta and TOP10 (negative control - Fig. 3.) genomic DNA
    Elongation time: 4 minutes
    20 cycles
  4. Gel electrophoresis of PCR products (there weren't any products for T7 RNA polymerase for translational fusion).

Fig. 1. Gradient PCR products for AID:
1 -DNA ladder; 2 to 13 -PCR products: In lane 2 is product of PCR reaction with annealing temperature 62°C (the lowest temperature of gradient), in lane 13 is product of PCR reaction with annealing temperature 82°C (the highest temperature of gradient). The lanes between them contain products of PCR with middle annealing temperatures. Fig. 2. Gradient PCR products: upper - for transcriptional fusion, lower - for translational fusion; 1-DNA ladder; 2 to 12-PCR products-border annealing temperatures of gradient: 62°C (lane 2) to 82°C (lane 12) Fig. 3. Gradient PCR products for negative control (Top10 genomic DNA): 1-DNA ladder; 2 to 6-PCR products-border annealing temperatures of gradient: 62°C (lane 2) to 82°C (lane 6).


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