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

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