From 2013.igem.org

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Reculturing Bacteria

  • The P. putida grew overnight, which proved that we had viable cells.
  • The bacteria was then re-streaked to single colonies and left to grow overnight in a 37C incubator.

Isolation of P. putida DNA

  • 8 1.5ml samples were isolated from each of the 2 broths that were prepared yesterday.
  • The first few steps of CTAB protocol was used to prep cells for DNA isolation;
  • The 8 samples were spun down at 10000 rpm for 5 mins.
  • The supernatant was discarded.
  • cells were then resuspended in 1ml of TE.
  • 740ul of this new mixture was transferred to 8 new eppendorf tubes.
  • 20ul of lysozyme was added to each of these 8 samples.
  • A maxwell robot was then used to complete the DNA extraction process.

Hydrodynamic shearing of Herring Sperm DNA

  • A 20G syringe was used ~24 times to squirt the herring sperm DNA in order to make it less viscous.
  • 30G syringe was also used
  • This method seems to work better than the sonification.