Why did I get no colonies when I cotransform the PmeI-cut shuttle plasmids with pAdEasy backbone vectors into BJ5183 cells?
In most cases, this is a result of bad competent cells. Make sure your BJ5183 cells are highly competent. Also, you should always include a positive control plasmid in your transformation. Another common cause of this problem is the quality of your miniprep DNAs. As mentioned in the protocol, the pAdEasy vectors should be CsCl-banded. It is very difficult to get good and consistent quality of the shuttle vector DNA from most commercial miniprep kits. A lot nicked DNA will be generated from these prep kits. In our experience, we get the most reliable and consistent results when the conventional alkaline lysis miniprep procedure (see Protocol section) is used. Majority of the miniprep DNA from this procedure are in supercoiled form, which is important for correct homologous recombination.
Related Questions
- Which strand of DNA is packaged when cells carrying the pMAL vectors are superinfected with an M13 helper phage such as M13KO7 (NEB cat #315)?
- Why did I get no colonies when I cotransform the PmeI-cut shuttle plasmids with pAdEasy backbone vectors into BJ5183 cells?
- How are cloning vectors and plasmids used in gene transfer experiments
