Hi all,
I am not getting any colonies when I transform One shot ccdB survival 2 T1 cells with ccdB vector with low concentration (2ng/reaction). I could not increase the concentration as I have very limited amount of the vector (only 5ng is left). The protocol says that ccdB survival 2 T1 cells can take up to 10pg of the vector. I am not sure what went wrong. Is there anyone, who can help in troubleshooting.
it is hard to troubleshoot because we don't really know the experiment you are doing. Do you have a pure plasmid stock you are transforming, or is this a cloning experiment?
But things to check are 1) are the competent cells good (do a test transformation with a control plasmid); 2) is your plasmid DNA good (harder to test if you only have 5 ng) but you could try to transform a tiny bit into a non-ccdB host; 3) your ligation didn't work for some reason.
Check the transformation frequency of the cells with a different plasmid. By the way, you do not need ccdB survival 2 T1R cells; any strain carrying an F episome will do. Also, are you using the correct antibiotic? If I were you I would increase the recovery step during transformation from 1 h at 37*C to static overnight incubation at room temperature.
Hope it helps!
Thank you one and all for the valuable suggestions. Michael J. Benedik : I am doing some promoter swapping study using the limited aliquot of ccdb vector which I am going to use as a plant expression vector. I already used up the pure plasmid stock for the cloning work (the left 5ng). We are now trying to source the pure plasmid stock. And we are preparing new competent cells too. This time we will try to transform into non ccdb cells too. Alejandro Martin : yeah the bacterial selection antibiotic is correct. Kanamycin and Chloramphenicol. Although I am using only kanamycin. Yes, this time I can increase the incubation period a bit longer. Hoping to get the pure plasmid vector soon
- Badges
- Science topic
- Similar topics
- Analytical Chemistry
- Column