User:Jeffrey Kim/Notebook/Halogenated natural products from uncultured bacteria/2009/01/15

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TAR cloning

  • Clones grown in 5ml O/N with 5ug/ml tet and 100ug/ml Amp from single colony picks of the transformed recombined capture plasmids. 2ul used for electroporation of commercial EC100's to test for efficiency. Several hundred colonies popped up but this may be a limiting step (homemade EC's may not pick up the plasmid, lower efficiency, etc.).
  • 500ul saved for Glycerol stock and the rest was Qiagen mprepped for analysis: RE map of clones versus regular construct.
  • EcoRI digest of the plasmid releases the appropriate sized fragments (see figure below) indicating 100% efficiency of capture vector construction using yeast. (this system is magical....)
  • Capture vector saved for downstream recombination and targeted construction of biosynthetic pathway.