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Transformation results

  • E. coli transforms at 400 colonies
  • No MF transformants on any plates
  • Frozen transformed stocks from A and B grew well on the same plates
  • C did not grow, nor did wt MF
  • yeast (or other contaminants) on two plates. This is likely from the ice bucket water.

Remaining speculations

  • wrong DNA sequence
  • too low a cell density
  • hold on ice for 10-30 minutes prior to electroporation
  • recovery wrong
    • recovery at low temperature (perhaps in the cuvette)
    • osmolarity of the recovery medium is wrong
  • Test higher osmolarity of electroporation buffer than wash medium
    • some used 0.5 M sorbitol and 0.5 M manitol, 10% glycerol
    • some buffers had Mg++, but we can't use it due to transposomes

DNA sequence analysis

  • PCR from A/B strains
  • PCR from transposome mix
  • PCR from transformed E. coli

Good controls

  • E. coli transformation
  • growth of MF tetR strain
  • measurement of CFU in transformed input