Team:Warsaw/Calendar-Main/27 August 2009

From 2009.igem.org

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<h3><div style="text-align: center;">Assembly of endosomal detection operon</div></h3>
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<h4>Marcin</h4>
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<br />
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<p>Task 1:</p>
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<ul>
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<li>Digest of following constructs:</li><ul>
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<li><a href="http://partsregistry.org/Part:BBa_C0051"><span style="color: black">BBa_C0051</a></span> with <a href="http://partsregistry.org/Part:BBa_B0032"><span style="color: black">BBa_B0032</a></span> on <a href="http://partsregistry.org/Part:pSB1A3"><span style="color: black">pSB1A3</a></span> plasmid</li>
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<li><a href="http://partsregistry.org/Part:BBa_E0022"><span style="color: black">BBa_E0022</a></span> with <a href="http://partsregistry.org/Part:BBa_B0032"><span style="color: black">BBa_B0032</a></span> on <a href="http://partsregistry.org/Part:pSB1A3"><span style="color: black">pSB1A3</a></span> plasmid</li>
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</ul>
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</ul>
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<p>Methods:</p><ul>
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<li>
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Reaction mixture composition:</li><pre>
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20 &mu;l purified plasmid DNA product
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1 &mu;l XbaI (Fermentas)
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1 &mu;l PstI (Fermentas) in the case of C0051
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0.7 &mu;l SpeI (Fermentas) in the case of E0022
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2 &mu;l Buffer Tango (Fermentas)
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15 &mu;l MQ water</pre>
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<li>Digest was performed about seven hours</li>
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<li>After digestion samples were frozen</li>
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Revision as of 22:48, 3 September 2009


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Cloning of the mgtc promoter into the pSB1A3 plasmid

Kamil


Tasks:

  • Amplification of the mgtc promoter using new primers
  • Isolation of the pSB1A3 plasmid

Methods:

  • The PCR mix was prepared as follows: 

    5μl buffer B (EURx)
    5μl 10mM dNTPs
    5μl forward starter
    5μl reverse starter
    2μl OptiTaq polymerase (EURx)
     2μl Salmonella matrix
     26 μl H2O
  • PCR programme: 

     4min 95°C 
     (30s 95°C, 35s 56°C, 40s 72°C)x28 
     10min 72°C 
     ~ 4°C
  • The PCR results were visualised with gel electrophoresis on 1% agarose gel.
  • The plasmid isolation was carried out from 3μl of culture cultivated for 6h in 37°C. The Plasmid Mini kit (A&A Biotechnology) was used.

Results:

  • Nothing

Conclusions:

  • The temperature was chosen... poorly.

Cloning of the cro-box into the pSB1A3 plasmid

Kamil


Tasks:

  • Creation of the cro-box double stranded DNA

Methods:

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