My transformation after Gibson cloning isn't working. I am using NEB-alpha cells which are good for transformation but even positive control that comes with Gibson kit didn't work on the chloramphenicol plates that I am plating my cells on.
I incubate cells at 37C and also at Room temperature (2 days incubation) for slower growth of plasmid but even that doesn't work (plasmids I have seem toxic for cells). I even reduced the amount of Chloramphenicol to allow cells to grow but I don't see any colonies. I plated cells on plain lb and they grew fine. What needs to be done?
My plasmid has dead-Cas9 in it and sgRNA components.
How should I improve the transformation? Thanks.
1) If the positive control is not working, maybe something is wrong with the protocol. Are you using the correct antibiotic concentration (Gibson suggested 33 µg/ml for chloramphenicol)?
2) Is your plasmid resistant to choramphenicol?
NEB's Gibson positive control is pUC19. It has ampicillin resistance not chloramphenicol.
I don't believe anyone commonly makes chloramphenicol Cas9 plasmids either. Why don't you double check that to make sure that your Cas9 doesn't actually have an amp or kan marker, as almost all Cas9 plamids out there do.
If your transformation and resistance schemes are working you should always see a large number of colonies, regardless of whether or not your assembly works. No colonies means something else is wrong, and you don't even get to test the assembly itself.
Thank you Wong Ling Chie and John Schloendorn for your very helpful comments. I think you both are right, it must be the wrong antibiotic that I am adding. I can double-check it with the lab member I got this plasmid from (I was told it has chlor resistance) and also look for resistance gene in plasmid sequence.
I double-checked the sequence and it does have chloramphenicol resistance gene. I will check the positive control too.
- Badges
- Science topic