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From 2014.igem.org

Protocols / 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. By making use of 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. Ligation product were transferred into E.coli and analyzed via colony PCR. Obtained plasmids were isolated, sequenced and sent to the iGEM Headquater.

Labwork

• Amplify the sequence of interest by a standard PCR
  • 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.
• All fragments, the purified PCR products and the isolated final vectors were cleaved.
  • by 20 µl digest mixes including the PstI and EcoRI enzymes.
  • While ~ 500 ng of plasmid DNA was sufficient, the highest possible volume of eluted insert DNA was used.
• Due to instable single-stranded overhangs of the digest products, analysis via agarose gel electrophoresis and purification via silica membranes were performed subsequently. For stable insertion the listed ligation mix (10 µl) was kept at RT for 1 h:

Ligation Mix

4.0 μl insert

3.0 μl vector

1.0 μl ligase buffer

0.2μl ligase

1.8 μl H2O

• Ligation products were transferred into E. coli.
• E. coli were grown on chloramphenicol containing 2TY medium and checked by colony PCR.