Team:Warsaw/Calendar-Main/27 August 2009

From 2009.igem.org

(Difference between revisions)
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<li>The intA gene that was used might not be the intA gene after all. </li>
<li>The intA gene that was used might not be the intA gene after all. </li>
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<br />
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<h3><div style="text-align: center;">Cloning switch 1 regulatory parts [
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<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K177012">K177012</a>-PcI.RBS.LacI,
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<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K177033">K177033</a>-PcI.RBS.LacI.PcI.RBS.RFP.terminator,
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<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K177011">K177011</a>-PLacI.RBS.cI.terminator,
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<a href="http://partsregistry.org/Part:BBa_K177038">K177038</a>-PLacI.RBS.cI.terminator.PLacI.RBS.GFP.terminator
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]
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into two compatible low copy number plasmids of different antibiotic resistance</div></h3>
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<h4>Ania</h4>
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<br/>
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<p>Tasks:</p>
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<ul>
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<li>
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</li>
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</ul>
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<br/>
</html>
</html>

Revision as of 00:17, 10 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:

  • The mixture of equal volumes (10μl) of both ssDNA was heated to 94°C for 15min. and left to cool.

Assembly of endosomal detection operon

Marcin


Task 1:

Methods:

  • Reaction mixture composition:
    •  
20 μl purified plasmid DNA product
1 μl XbaI (Fermentas)
1 μl PstI (Fermentas) in the case of C0051
0.7 μl SpeI (Fermentas) in the case of E0022
2 μl Buffer Tango (Fermentas)
15 μl MQ water
  • Digest was performed about seven hours
  • After digestion samples were frozen

Making of the plac-RBS-llo-intA part

Jarek


Tasks:

  • Separation of digested DNA fragments in 0,8% agarose.

Results:

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