User:Tamra L. Fisher/Notebook/Research for Dr. Hartings/2012/05/21

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Transformation Plates

Because I left the transformation plates on the counter for the rest of the weekend at room temp there was a potential for growth, and colonies did appear, but it is unlikely that these are the transformation products from Friday. I will grow them in 3mL of LB with 3μL of 100mg/mL ampicillin at 37°C at 250rpm for about six hours to see if they are ampicillin-resistant colonies.

Preparation of Buffers

  • 1L 25mM Tris, 50mM NaCl, pH 8: 3.03g Tris + 2.92g Nacl + dH2O + HCl (to pH)
    • Actual pH was 7.99
    • This buffer was filtered

Filtering Protein

  • The triple mutant that was expressed last week, and the double mutant (expressed in April) were both vacuum filtered today.

Test Transformation

A transformation with the test plasmid that Novagen sends with its competent cells to see if the transformation problem can be narrowed down.

  1. Take a sterile microcentrifuge tube and place it on ice.
  2. Mix 50μL of NovaBlue Competent E.coli with 5μL of Novagen 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 or PCR product.
  6. Shake the mixture at 250rpm at 37°C for one hour.
  7. Spread 100μL of the mixture on an LB plate with 100μg/mL ampicillin.
    • The LB plates were made with 0.88g LB, 0.7g Agar, and 35mL of distilled water. The mixture was autoclaved on a liquid cycle. When the mixture cooled to about 60°C 35μL of 100mg/mL ampicillin was added to it and it was poured into a sterile petri dish.
  8. Incubate the plate overnight (inverted) at 37°C.