I have a pDonr221 vector, that when sequenced clearly has the ccdB (Cell death gene) - so normally would only be able to propigated in cell death resistant E.coli strains. Recently, when I do a BP reaction, my colonies seem to be JUST the origional pDonr221 vector- and no insert. Also, when I do a negative control transformation side by side with my BP, I get more colonies on that plate then the BP reaction.
I am using homemade competent DH10B cells, but I also tried this transformation with control in Stellar Cells (that come with the inFusion kit) = same result.
I haven't sequenced my colones yet, I'm hoping some will be positive for my insert from the BP reaction. But has anyone experienced this? I can only think that either the cells are able to transform the ccdb gene, or something is wrong with the plasmid? But when it is sequenced it clearly shows the correct sequence.
Please let me know your thoughts or experience.
Thank you!
- Badges
- Science topic
- Similar topics
- Geoscience
- Asia
- India