Hi Dear All,
For the past few weeks, I was trying to insert Hygromycin resistance cassette (eukaryotic expression) into a lentiviral vector.
I successfully inserted the sequence (~1.4kb) into a 7.7kb vector both by overlap extension PCR and by restriction enzyme cloning as I confirmed ligation/Overlap extension PCR by amplifying insert from recombinant E.coli DH5a colonies by colony PCR.
I verified recombination by amplifying insert as well as plasmid-insert junction sequence by colony PCR
Everything goes well until colony PCR but after that I lose recombinant plasmid and only get recipient vector from minipreps.
Why do I lose recombinant plasmid upon culturing DH5a cells? Is the hygromycin resistance cassette (HSV TK promoter-HygromycinR-HSV TK poly A signal) toxic to DH5a cells?
I inserted Hygromycin resistant cassette at different positions by both methods without interfering with Origin of replication or ampicillinR region.
Desperately looking forward to hear any positive feedback.