Team:Warsaw/Calendar-Main/13 May 2008

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<html><h3>Preparation of pMPMT5+AID construct and PCRs for fusions</h3>
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<html><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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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/><h3>1-DNA ladder; 2 to 13-PCR products (2-annealing temperature 62&deg;C, 13-annealing temperature 82&deg;C)</h3>
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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>
<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>
<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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<img src="https://static.igem.org/mediawiki/2008/9/97/T7grad_WAW1.jpg" width=350/>
<img src="https://static.igem.org/mediawiki/2008/9/97/T7grad_WAW1.jpg" width=350/>
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<h3>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.</h3>
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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>
<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 - negative control (Top10 genomic DNA). 2-annealing temperature 62&deg;C, 6-annealing temperature 82&deg;C</h3>
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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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</p>

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

  1. Setup of 8 separate cultures from 8 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).
    Primers: AIDlNrH AIDpLinB
    Template DNA: pTrc99A-AID
    Elongation time: 60 sec
    20 cycles
    3. 1-DNA ladder; 2 to 13-PCR products (2-annealing temperature 62°C, 13-annealing temperature 82°C)
  3. Optimization of conditions for PCR - T7 RNA polymerase for translation fusion; annealing temperature gradient (from 62°C to 82°C).
    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).
    Primers: T7lRBSHi T7pXbSal
    Template DNA: E. coli Rosetta and TOP10 (negative control) 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).
PCR products: upper - for transcription fusion, lower - for translation fusion; 1-DNA ladder; 2-annealing temperature 62°C; 12-annealing temperature 82°C. PCR products - negative control (Top10 genomic DNA). 2-annealing temperature 62°C, 6-annealing temperature 82°C


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