User:Jeffrey Kim/Notebook/Halogenated natural products from uncultured bacteria/2009/01/08

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TAR cloning progress

  • miniprepped plasmids (pLLX8 clone ZB118) were used at 1ul (200ng) for PCR with Buffer G and failsafe hi fidelity enzyme. 25 cycles at 65C (30' extension time) yielded bands of the appropriate sizes (1kb, 2.1kb) for ZB118.
  • Extension time for pLLX8 amplicon (2.6 kb) may have been too short, very faint product although of the correct size.
  • Ran a gradient with buffer G with an extension time of 3minutes for 25 cycles. Temps 55-62.5C looked good (annealing temp) and these products were combined 25ul rxn, and minelute purified in parallel with the ZB118 amlicons for US and DS targeting sequences.