User:Tamra L. Fisher/Notebook/Research for Dr. Hartings/2012/05/24

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Harvesting Cells

  1. Centrifuge the cells expressed overnight at 4500rpm at 4°C for 15 minutes.
  2. Resuspend the pellets in 25mM Tris 50mM NaCl, pH 7.99 (sterile-filtered).
  3. Place resuspended cells in -20°C freezer.

Note: The wild-type pellets had a pinkish color to them and the triple mutant pellets were only a tannish-color.

Test Transformations

I'm trying two more transformations to try to see if I can narrow down the problem with transformations lately. I'll be trying the transformations with the competent cells made on 11/8/11 and 11/15/11 (I've only been using the 11/8/11 cells because they were determined to be more competent previously. Also, for both of the transformations I will use the SOC provided by Novagen to see if the student-made SOC was affecting the transformations.

  1. Take a sterile microcentrifuge tube and place it on ice.
  2. Mix 50μL of NovaBlue Competent E.coli with 5μL of the test plasmid in the cold, sterile tube.
  3. Incubate this mixture for 30 minutes on ice.
  4. Transfer the tube to a heat block at 42°C for 90 seconds, and then transfer the tube back to ice for 5 minutes.
  5. Add 200μL of SOC media to the cells/plasmid.
  6. Shake the mixture at 165rpm at 37°C for one hour.
  7. Spread 100μL of the mixture on an LB plate with 100μg/mL ampicillin.
  8. Incubate the plate overnight (inverted) at 37°C.