From 2013.igem.org

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Measuring the concentrations of isolated P. putida DNA from the previous day

  • Measuring concentration using nanodrop
  • Blanking against buffer
Sample nr.Concentration ng/ul260/280260/230

Preparing samples to run on 1% gel

  • 500ng of each of the isolated P. putida DNA sample is run on gel
  • Volumes are adjusted to 20ul per well accordingly (includes DNA, sterile H2O and OG)
  • 5ul of orange G dye is added to each sample
  • 5ul of 1kb ladders are used
  • For sheared DNA, dilutions were loaded
    • 1ul of DNA was diluted in 9ul of 1xTE buffer to make up 10x dilution
    • 1ul from 10x dilution was put in 9ul of 1xTE buffer to make up 100x dilution
  • Dilutions were nanodropped and the concentration for 100 fold dilution was 121 ng/ul
  • 200ng of herring sperm DNA was run on gel
  • One lane was left for control, non-sheared DNA, also 100 fold dilution

Running gel of P. putida DNA and Herring sperm DNA

Igem putida 220813.jpg

Samples in lanes goes as follows: 1 Kb ladder, 2, 4, 6, 8, 10, 12, 14, 16, 1 kb ladder, 100x dilution of sheared herring sperm DNA, control 100x diluted herring sperm DNA

Running sonicated herring sperm DNA on 0.8% gel

  • 3 lanes for running:
    • Sheared and sonicated
    • Unsheared and sonicated
    • Unsheared and unsonicated
  • 5ul of 1kb marker is used
  • The DNA samples are diluted 100 fold and 200ng of sample is run.
  • Volume of sterile water is adjusted appropriately to make up 20ul of sample per well, including 5ul of orange G dye

Igem shear son 220813.jpg

  • Not visible on the computer file, but picture shows a very slight decrease in sheared/sonificated and unsheared/sonicated samples vs the unsheared/unsonificated control
  • The difference is not significant, but the viscosity has changed when mechanically spreading the DNA on polystyrene, though it is not that evident on the gel.

Making up a sheared/sonicated dilution to leave on bench overnight

  • 500ul of sheared/sonicated DNA
  • 500ul of 1xTE
  • Mix
  • Leave on bench overnight

Incubating sheared/sonicated dilution at 37C on a shaking rack

  • 10ul of 1xTE added to 2ul of sheared/sonicated DNA
  • Mix
  • Incubated overnight at 37C on a shaking rack