User:Tamra L. Fisher/Notebook/Research for Dr. Hartings/2012/07/06

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Reconstitute Asc Hb with Nickel

  • The stir plate was turned off at the beginning of the day, so the protein/Ni(protoporphyrin IX) mixture could sit undisturbed.
  • Six and a half hours later, the protein was poured into centrifuge tubes and spun at 18000rpm for one hour at 4°C.
  • The supernatent was then poured into dialysis tubing and placed in 25mM Tris, 50mM NaCl, pH 8 buffer overnight (with stirring).
    • The pellet did break up into the supernatent a bit so it should pobably be re-spun tomorrow and should then be decanted from the centrifuge tube more carefully.

Transformations

Using the cells that were made competent yesterday, the transformation procedure from the same paper the competent cell procedure was based on ("High Efficiency Transformation of Escherichia coli with plasmids" Inoue et. al.) will be followed.

  1. Chill the plastic culture tubes on ice for 15 minutes.
  2. Add 200μL of cells to each tube.
    • the BL21(DE3) and NovaBlue strains were used today
  3. Add 3μL of DNA to each tube and mix by pipetting up and down.
    • the pET3d plasmid was used as a positive control to transform each cell strain, and the wildtype ligation product from 07/04/12 was also used to transform both strains.
  4. The cells with DNA sat on ice for 30 minutes.
  5. The cells were then heat shocked at 42°C for 30 seconds and then placed back on ice.
  6. 0.8mL of SOC was added to each tube and the cells shook at 250rpm for one hour at 37°C.
  7. 100μL of cells from each tube was plated on an LB/Amp plate (ampicillin concentration = 100μg/mL).
  8. The plates were incubated overnight at 37°C.



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