Team:Warsaw/Calendar-Main/13 May 2008

From 2008.igem.org

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<li>Setup of 8 separate cultures from colonies of transformants pMPMT5+AID (liquid LB+tetracycline).</li>
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<li>Setup of separate cultures from colonies of transformants pMPMT5+AID (liquid LB+tetracycline).</li>
<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). Fig. 1.<br>
<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). Fig. 1.<br>
Primers:
Primers:
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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 transcription fusion; annealing temperature gradient (from 62&deg;C to 82&deg;C). Fig. 2. <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 Fig. 3.) genomic DNA<br>
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20 cycles <br>
20 cycles <br>
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<img src="https://static.igem.org/mediawiki/2008/9/97/T7grad_WAW1.jpg" width=350/>
 
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<var>Gradient PCR products: upper - for transcription fusion, lower - for translation 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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<img src="https://static.igem.org/mediawiki/2008/7/71/Aid_pcr_grad_WAW.jpg" width=350/><var>Fig. 1. Gradient PCR products for AID: 1-DNA ladder; 2 to 13-PCR products-border annealing temperatures of gradient: 62&deg;C (lane 2) to 82&deg;C (lane 13)</var>
<img src="https://static.igem.org/mediawiki/2008/7/71/Aid_pcr_grad_WAW.jpg" width=350/><var>Fig. 1. Gradient PCR products for AID: 1-DNA ladder; 2 to 13-PCR products-border annealing temperatures of gradient: 62&deg;C (lane 2) to 82&deg;C (lane 13)</var>
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<img src="https://static.igem.org/mediawiki/2008/9/97/T7grad_WAW1.jpg" width=350/>
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<var>Fig. 2. Gradient PCR products: upper - for transcription fusion, lower - for translation 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>
<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/>
<var>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&deg;C (lane 2) to 82&deg;C (lane 6).</var>
<var>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&deg;C (lane 2) to 82&deg;C (lane 6).</var>
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Revision as of 09:06, 11 October 2008

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Preparation of pMPMT5+AID construct and PCRs for fusions

Michał K.

  1. Setup of separate cultures from colonies of transformants pMPMT5+AID (liquid LB+tetracycline).
  2. 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
  3. Optimization of conditions for PCR - T7 RNA polymerase for translation 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
  4. Optimization of conditions for PCR - T7 RNA polymerase for transcription 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
  5. Gel electrophoresis of PCR products (there wasn't any products for T7 RNA polymerase for translation fusion).

Fig. 1. Gradient PCR products for AID: 1-DNA ladder; 2 to 13-PCR products-border annealing temperatures of gradient: 62°C (lane 2) to 82°C (lane 13) Fig. 2. Gradient PCR products: upper - for transcription fusion, lower - for translation 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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