Can I use LB and RB boarder primers to amplify the either sides of the trans-gene (cassette)? For this does it necessary to check the copy number of the transgene first?
see this link
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4664744/
Do you already have an estimated location? If so, design primers to flank your potential insert site and use the LB primer. The right border is often lost during the insertion and/or you can have concatenated insertions. Then sequence your products. Otherwise, you will have to use degenerate primers and "TAIL" PCR to find your insertion site.
It's VERY to have insertions or deletions of a few hundred base pairs of DNA at the insertion site. It's also VERY common (~20%) to have a chromosomal translocation associated with your T-DNA insertion. Bottom line: you can only find them by genetic mapping and you can't do anything about them. Message me if you want to know more about T-DNA lines in Arabidopsis
Article Chromosomal translocations are a common phenomenon in Arabid...
Thank you Dr. Katie,
what do you mean by the "estimated location"? and how can I know this fact?
- Badges
- Science method