I am working with some MC1061 cells that were transformed with my plasmid of interest a while back. Yesterday morning I took a swab from our cryo-store and streaked an agar plate with 10ug/ml Tetracycline and 15ug/ml Ampicillin. What is the growth time for this strain of cell? I have colonies at 24 hours post inoculation, but the colonies are small. What is a good rule of thumb for the growth of these cells?
You should be plating them on Kanamycin medium. The Amp and Tet resistant markers both carry amber mutations in them which are suppressed when you transform with a supF (suIII) plasmid. In the absence of that plasmid MC1061/P3 should be sensitive to Amp and Tet. The P3 plasmid is maintained by kanamycin.
Our plasmid has an Amp gene. This is the reason we are doing the Amp and Tet, not the Kan. Our plasmid has the pCDM8 backbone.
Sorry I misread your original post. However the cells should still be Kan-R which is the marker for the P3 plasmid. I would test your stock out on each of the 3 antibiotics separately just to get an idea of what the problem might be. I would think you should have very healthy colonies by 24 hours.
You should regrowth E.coli again because E.coli in freeze that was damage from ice. If you regrowth again in broth before spread on agar is a better forcolony size.
- Badges
- Science topic
- Similar topics
- Biological Science
- Biochemistry
- Biochemical Techniques