IGEM:Cambridge/2008/Notebook/Magnetic Bacteria/2008/07/31

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Getting MagA Out

Transformation of TOP10 for MagA and I0500

  • No colonies formed on the Kanamycin agar plates as we failed to transform cells
  • Transformed 50μL competent TOP10 with 1μL MagA plasmid again
  • Transformed 150μL CaCl2 treated TOP10 in LB broth with I0500 plasmid and 50μL of such cells with pUC plasmid as control
  • MagA transformed TOP10 plated on Ampicillin, Chloramphemicol, Tetracyclin and Kanamycin plates in 1/10 dilution to check plasmid resistance
  • pUC control and I0500 TOP10 on Kanamycin plates
  • All plates incubated at 37°C overnight
  • Mag A PCRed with new MAGA programme on the cool black PCR machine :)
    • 10 cycles in touchdown at 47°C
    • 20 cycles with 78°C annealing temperature
  • PCR product run on 1% agarose gel with SyBr Green
    • Lane 3 - 100bp ladder (1μL ladder + 7μL TAE + 2μL dye)
    • Lanes 5 and 7 - PCR-ed MagA (5μL DNA + 15μL SDW + 4μL dye)

Result:

  • Bands at around 1.3kb on lanes 5 and 7 corresponding to the size of MagA
  • Smear of DNA along the two lanes
  • Looked like genomic DNA on gel
  • Suspected having too much DNA on gel and suggested to do single colony PCR

Contact L.E. Bertani at Caltech for MagA gene map!!!



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