Difference between revisions of "Team:Goettingen/Experiments"

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     <li>Incubate plates at 37<sup>o</sup>C for minimum 3-4 days (timing can be extended depending on bacterial strain).</li>
 
     <li>Incubate plates at 37<sup>o</sup>C for minimum 3-4 days (timing can be extended depending on bacterial strain).</li>
 
     <li>After 3-4 days look for plates having clear zone of activity around cellulose substrate.</li>
 
     <li>After 3-4 days look for plates having clear zone of activity around cellulose substrate.</li>
 +
<p>&nbsp;</p>
 +
<p style="text-align: center;"><span style="font-size: x-large; color: #888888;"><strong><span lang="EN-GB">Esterase Activity plates, with 1% Tributyrin</span></strong></span></p>
 +
<p><span lang="EN-GB">&nbsp;</span></p>
 +
<p><span lang="EN-GB">To 500ml of LB Media add 7,5g of Agar and 5ml of Tributyrin and homogenize with a mixer.</span></p>
 +
<p><span lang="EN-GB">This culture medium must be directly sterilized by autoclaving at 121˚C for 20 min.</span></p>
 +
<p><span lang="EN-GB">If you wait too long it will be inhomogeneous again!</span></p>
 +
<p><span lang="EN-GB">When the medium cools down enough, the antibiotic can be added.</span></p>
 +
<p><span lang="EN-GB">&nbsp;</span></p>
 +
<p><strong><span lang="EN-GB">Result:</span></strong><span lang="EN-GB"> Halo formation is visible around the positive clones.</span></p>
 
</ul>
 
</ul>
  

Revision as of 09:15, 14 September 2015



LB Medium

Plasmid Extraction - using peqGOLD Plasmid Miniprep Kit I (PEQLAB Technologies)

Plasmid Extraction - using QIAprep Spin Miniprep Kit (QIAGEN)

Blunt End Ligation in pJET1.2 vector –Clone JET PCR Cloning Kit–Thermo Scientific

TOPO® Cloning protocol usingChampion™ pET Directional TOPO® Expression Kits. Thermo Fisher Scientific

PCR product purification using QIAquick® PCR Purification Kit (QIAGEN)

Plasmid transformation into chemically competent E. coli

Preparation of competent E.coli cells

Esterase activity test

PCR Gel extraction, peqGOLD Gel Extraction Kit

Electroporation of BL21 cells with pJET_RFP

Media preparation for cellulase activity screening

Cellulase activity screening

 

  • Streak Competent Top10 E. coli cells or Competent BL21 cells transformed with appropriate vector containing cellulase construct onto agar plates containing cellulose substrate.
  • Incubate plates at 37oC for minimum 3-4 days (timing can be extended depending on bacterial strain).
  • After 3-4 days look for plates having clear zone of activity around cellulose substrate.
  •  

    Esterase Activity plates, with 1% Tributyrin

     

    To 500ml of LB Media add 7,5g of Agar and 5ml of Tributyrin and homogenize with a mixer.

    This culture medium must be directly sterilized by autoclaving at 121˚C for 20 min.

    If you wait too long it will be inhomogeneous again!

    When the medium cools down enough, the antibiotic can be added.

     

    Result: Halo formation is visible around the positive clones.

Experiments & Protocols

Describe the experiments, research and protocols you used in your iGEM project.

What should this page contain?
  • Protocols
  • Experiments
  • Documentation of the development of your project

Inspiration