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
- The triple mutant that was expressed last week, and the double mutant (expressed in April) were both vacuum filtered today.
A transformation with the test plasmid that Novagen sends with its competent cells to see if the transformation problem can be narrowed down.
- Take a sterile microcentrifuge tube and place it on ice.
- Mix 50μL of NovaBlue Competent E.coli with 5μL of Novagen Test Plasmid in the cold, sterile tube.
- Incubate this mixture for 30 minutes on ice.
- Transfer the tube to a heat block at 42°C for 90 seconds, and then transfer the tube back to ice for 5 minutes.
- Add 200μL of SOC media to the cells/plasmid or PCR product.
- Shake the mixture at 250rpm at 37°C for one hour.
- 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.
- Incubate the plate overnight (inverted) at 37°C.