I would like to clone a 7bkp insert into a pUC18 vector (2,7kbp). Do you know if it may be somehow problematic? I mean ligation or transformation efficiency, insert stability or any other problems. The insert is a 100bp tandem repeat.
Gastón Paris
Yes, ligation and trasnformation efficiency drop as the insert size increase. I suggest to be sure to have enough ADN insert in order to have a 3:1 ratio (insert:vector) concentration. In a 7 kpb insert that means to have at least six times more ng of ADN than the vector. So, if you use 50 ng of vector you need to add 300 ng of insert. In addition use high efficient competent cell.
Another potential problem is the tandem repeat. Be sure to use RecA- E.coli cells.
2 votes
2 thanks
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Molecular Methods
- Cloning
More Dawid Deneka's questions See All
Similar questions and discussions