Also I have pET28a(+) frustrations
In order to express several fungal heat shock proteins I ordered BL21(DE3) E. coli cells and the pET28a(+) expression vector, both from Novagen. The fungal genes were synthesized by Baseclear and delivered as BamHI/XhoI fragments in the pUC57 plasmid. When I digest the pUC57 plasmids containing the fungal genes with BamHI/XhoI I get perfect bands of the correct size. When I digest the pET28a(+) expression vector I get also a nice band of the correct size. However, also a few faint smaller fragments were obtained. Serial digestion with BamHI and XhoI resulted in only the linearized pET28a(+) vector without the faint smaller fragments. After both the double and serial digestion I used Shrimp Alkaline Phosphatse. After ligation with the genes by using the Novagen DNA Ligation Kit and transformation, no colonies were observed while for the control (pET28a(+)) numerous colonies could be discerned. What am I doing wrong? 🙁