I am trying to extract Akata BAC from AGS-Bx1 cells and transformed into E. coli DH10B. EBV genes can be detected by qPCR with extacted product. But we cannot picked any EBV-positive DH10B colonies. If Somebody have a protocol to extract the bacmid from cells?
Are you using electrocompetent DH10B? Chemical transformation is very inefficient with large plasmid.
Yes，electrocompetent DH10B was used and it works good when electrotransformed with BAC extracted from E. coli instead of AGS-Bx1 cells. So I think the quality of BACMID extracted from AGS-Bx1 cells may be the key cause. Are there any suggestions about how best to tackle this problem? Thank you very much.
BACMIDS are low copy number plasmids so tend not to express antibiotic resistance marker proteins at the same levels as high copy number plasmids. Have you tried on a slightly lower concentration of antibiotic?
- Badges
- Science topic
- Similar topics
- Biological Science
- Microbiology
- Virology
- Virus