Hi all!

I have been trying for a while to clone two fragments in a plasmid for expression of long hairpin dsRNA. My strategy was to insert the sense fragment first and then the antisense on the same direction. Primers are designed in a way to have a linker at the end and formation of loop in lhRNA. I cloned the sense strand successfully but it's been a while I cannot insert the second fragment. Although all steps including amplification of the fragment, digestion of plasmid/insert. ligation and transformation are well set-up. May I ask if anyone has similar experience and can give me some advice on dsRNA gene cloning? Is there any possibility that plasmids containing lhRNA coding genes are unstable in E. coli? Thanks in advance.