IGEM:Harvard/2007/Kevin's 91r Notebook

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  • Step 1: Overnight culture from Perry's frozen stock of bacteria with LppOmpA plasmid.
  • Step 2: Plasmid prep of LppOmpA plasmid.
  • Step 3: Purification of plasmid.
  • Step 4: Design primers for PCR.
  • Step 5: Produce PCR product and run on gel.

Result: Good bands for all three reactions, but signs of template present in all three.

  • Step 6: Topo cloning reaction: Mix PCR product and pTrcHis2-TOPO
  • Step 7: Transform mix into OneShot E.coli cells
  • Step 8: Overnight culture of colonies to analyze positive clones
  • Step 9: Plasmid prep of culture
  • Step 10: Digestion with Nco1 and EcoR1 and run on gel.

Result: Colonies grown because of template. They either cut once (one site present in template) or cut twice (two close sites present on the template). Solution: Clonewell PCR product and select for the cut fragments.

  • Step 11: Clonewell PCR product
  • Step 12: Repeat steps 6-10.
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