IGEM:Cambridge/2008/Notebook/Magnetic Bacteria/2008/08/01

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Battle with MagA continues...

MagA and I0500 Transformation Plates

  • MagA transformants grew on Ampicillin plates and Kanamycin plates but not Tetracyclin and Chloramphenicol
  • I0500 transformants grew on Kanamycin plates
  • pUC control also grew on Kanamycin plates and that's not good!
  • Suspected problems with preparation of our own Kanamycin plates and so re-do MagA, I0500 and pUC TOP10 transformants on Kanamycin plates prepared by Ellis
  • Plates incubated at 37°C overnight

Single Colony PCR of MagA from Ampicillin Plate

  • Without using master mix but normal protocol
  • Using updated PCR programme MAGA2
    • Touchdown: 6 cycles; Annealing max:56°C min:46°C
    • Normal: 28 cycles; annealing at 76°C
  • Reagents added in this order:
    • 12.4μL SDW
    • 4μL 5xHF Phusion Buffer
    • 0.4μL dNTPs (10mM)
    • 1μL primer MagAF (10mM)
    • 1μL primer MagAR (10mM)
    • 1μL DNA
    • 0.2μL Phusion DNA polymerase
  • Run on 0.8% agarose gel with SyBr Green
  • Colony 4 has a faint band at 1.3kb for MagA
  • Quickly run colonies 1 and 5 on E-gel and re-confirm the absence of MagA in these 2 colonies but not the fault of agarose gel
Gel showing possible MagA PCR product at around 1.3kb from colony 4