Team:Hannover/Protocols/Cloning/Parts

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Results / Cloning the Shipping Vectors

We wanted to contribute useful Biobricks to the iGEM Parts Registry and thus cloned our important constructs into the iGEM pSB1C3 standard vector. In accordance with classical cloning, we amplified our sequences of interest, cleaved them with restriction endonucleases generating the same single-stranded overhangs as for the final vector backbone and ligated both parts finally. After the ligation products were transferred into E.coli and analyzed via colony PCR, the resulted plasmids were isolated and sent to the iGEM Headquater.

Labwork

  • Amplify the sequence of interest by a standard PCR
    1. for all insert, the metal-binding-sequence (TA=65 °C), the expansin as well as the Chlostridium CBD (both TA=60 °C) the pORE-E3_2x35S_T4MBP served as PCR templates. An elongation step of 0:45 min was used.
  • For the removal of remaining polymerase, the PCR products were separated via agarose gel electrophorese and purified via silica membranes. For yield optimization, the smallest possible elution volume was used.