Team:Warsaw/Calendar-Main/15 July 2008

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<h3> Preparation of alpha+A conctruct</h3><h4>Antoni</h4>
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<p><ol><li>Gradient <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#pcr">PCR</a> on alpha+A PCR products with <a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#AlphaL+SacI">AlphaL+SacI</a> and <a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#AP+NotI">AP+NotI</a> primers.</li>
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<li>Gel electrophoresis. Again without satisfying results.
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<li>Third <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#pcr">PCR</a> on alpha+A. This time two temperatures of annealing (68&deg;C and 72&deg;C) and gradient of DMSO.</li>
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<li>Gel electrophoresis (<a href="https://2008.igem.org/Team:Warsaw/Calendar-Main/15_July_2008#fig1">Fig. 1</a>).
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<li><a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#DNA_isolation_from_agarose_gel">Gel-out</a> of proper 1000 bp band.</li>
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</li></ol></p>
<a name="fig1"><img src="https://static.igem.org/mediawiki/2008/c/c5/PCRalfa%2BAzDMSO.jpg"></a>
<a name="fig1"><img src="https://static.igem.org/mediawiki/2008/c/c5/PCRalfa%2BAzDMSO.jpg"></a>
<var><b>Fig. 1.</b>Gradient PCR on alpha+A products. Lanes 1-3, 5, 6 - annealing temperature 68&deg;C and DMSO concentration of 2%, 4%, 6%, 8% and 10% respectively. Lanes 7-11 - 72&deg;C and DMSO concentration increasing in the same maner. Lane 4 and 12 GeneRuler DNA Ladder Mix 1 &mu;g and 0.5 &mu;g respectively.   
<var><b>Fig. 1.</b>Gradient PCR on alpha+A products. Lanes 1-3, 5, 6 - annealing temperature 68&deg;C and DMSO concentration of 2%, 4%, 6%, 8% and 10% respectively. Lanes 7-11 - 72&deg;C and DMSO concentration increasing in the same maner. Lane 4 and 12 GeneRuler DNA Ladder Mix 1 &mu;g and 0.5 &mu;g respectively.   

Revision as of 17:47, 26 October 2008

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Cloning of protein Z DNA to OmpA constructs

Michał K.

Two colonies (pACYC177+OmpA_Z_omega) was inoculated to liquid LB with kanamycin.

Preparation of alpha+A conctruct

Antoni

  1. Gradient PCR on alpha+A PCR products with AlphaL+SacI and AP+NotI primers.
  2. Gel electrophoresis. Again without satisfying results.
  3. Third PCR on alpha+A. This time two temperatures of annealing (68°C and 72°C) and gradient of DMSO.
  4. Gel electrophoresis (Fig. 1).
  5. Gel-out of proper 1000 bp band.

Cloning omega-A fusion on pKS (second attempt)

Michał L., Ewa, Marcin

Polymerase Chain Ligation on linker-A and omega-linker

  • reisolated PCR product omega-linker - 4 µl
  • reisolated PCR product linker-A - 13.5 µl
  • primer OmegaL+SacI - 2 µl
  • primer AP+NotI - 2 µl
  • Pfu buffer with Mg2+ - 5 µl
  • dNTPs - 1 µl
  • H2o - 22 µl
  • Program:
    1. 95°C - 3'
    2. 95°C - 30"
    3. 55°C - 45"
    4. 68°C - 1'
    5. go to step 2 25 x
    6. 68° - 10'
    7. keep in 4°
  • gel electrophoresis of products


Preparation of alpha+A conctruct

Antoni

  1. Gradient PCR on alpha+A PCR products with AlphaL+SacI and AP+NotI primers.
  2. Gel electrophoresis. Again without satisfying results.
  3. Third PCR on alpha+A. This time two temperatures of annealing (68°C and 72°C) and gradient of DMSO.
  4. Gel electrophoresis (Fig. 1).
  5. Gel-out of proper 1000 bp band.

Fig. 1.Gradient PCR on alpha+A products. Lanes 1-3, 5, 6 - annealing temperature 68°C and DMSO concentration of 2%, 4%, 6%, 8% and 10% respectively. Lanes 7-11 - 72°C and DMSO concentration increasing in the same maner. Lane 4 and 12 GeneRuler DNA Ladder Mix 1 μg and 0.5 μg respectively.

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