I trying to clone TOP3B in pAC5.1/V5-His vector and express the vector with insert later in Drosophila cells. But when I try to clone TOP3B in pAC5.1/V5-His vector in DH5alpha cells, none of the colonies on the transformation plate are positive for the TOP3B insert. My cloning protocol has worked for the insertion of all other kinds of genes into pAC5.1/V5-His vector in DH5alpha cells, but just not TOP3B. So, I am thinking maybe there is a leaky expression of pAC5.1/V5-His vector with TOP3B insert in DH5alpha cells, which can be toxic to DH5alpha. How do I prevent this leaky expression of pAC5.1/V5-His vector with TOP3B insert in DH5alpha cells? Are there other competent cells where the pAC5.1/V5-His vector will not be expressed? Ideally pAC5.1/V5-His vector is not supposed to have any bacterial promoter. I don't know why an e coli polymerase is driving its expression.
How are you checking for the presence/absence of the TOP3B insert?
Colony PCR, restriction digest, etc?
Hi Sushmita,
if your problem is really toxicity due to leaky expression you should first try to reduce your temperature while growing your colonies (30-20°C). This can already help. However there are also certain E.coli strains which are more suitable for growing toxic genes like 5-alpha F'Iq Competent E. coli from NEB (having a tighter expression control) or CopyCutter™ EPI400™ E. coli* from Biosearch Technologies(reducing the copy number of plasmids).
- Badges
- Science topic
- Similar topics
- Physical Sciences
- Materials Science
- Materials Chemistry
- Polymer Chemistry