Dear all,
I am currently preparing a probe for whole-mount in-situ hybridization. I have been given constructs (that are validated by sequencing) and ordered to carry out a transformation. After the transformation, I carried out plasmid kit extraction and realized that most of my plasmids seem to be nicked. Neither did I carry out vortexing during the whole process nor did I allow the P2 (FADP2) Buffer to lyse my cell for too long (I had lysed them for 3 minutes and immediately added the P3 Buffer for neutralization). May I ask whether this is potentially caused by:
(1) Using colonies that are not freshly prepared. Mine was prepared two days ago and stored in a 4 degree Celcius fridge.
(2) Overgrown of Bacteria Culture. I have been shaking my 4.5ml Broth + 4.5 1000x µl Amplicilin for 20 hours. I know that the optimal time for that is 14~18 hours but I really have no choice at that time.
I would also like to ask whether my plasmids being nicked will result in their inability to linearize or it simply will work as to how supercoiled plasmids do?
Don't worry about it. If you intend to linearize then it will be fine and they will cut just fine. It is only an issue if your protocol requires supercoiled plasmid as template.
However I wonder whether in fact your plasmid is nicked, what is the criteria you are using to determine that? You might run some uncut plasmid alongside a cut sample (single cleavage) and see how the gel looks.
A nicked plasmid will run in a gel with about the same mobility as the line arises plasmid, so running a digested contril alongside is informative. It is also possible that you have supercoiled dimer plasmid . These will all resolve upon digestion.
- Badges
- Science topic