I received synthesized genes from Genescript in pUC57 vector two months ago. Since then, I've tried unsuccessfully to subclone them into pET28 expression vector. This is our protocol:
1.Amplify insert DNA using specific primers with flanking NdeI and XhoI restriction sequences
2. gel-purify insert to separate it from pUC57
3. digest product and pET28a with NdeI and XhoI
4. ligate pET28 and insert with T4ligase (22oC 1hr or 4oC overnight); 1:3, 1:1, 1:5 vector:insert ratio.
5. Transform into E.coli Top10cells
Each time I extract the plasmid and digest with NdeI and XhoI, the insert is not there.
I've tried several times including digesting directly from pUC57 (EcoRI/XhoI) and transforming into JM109 cells to no avail. We use thermoscientific fast digest enzymes in my lab which usually involve 5mins digestions. I extend this to 45mins/1hr, yet, no insert.
Pls, does anyone have any useful suggestions regarding this for me?