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pET28a(+) frustrations

Discussion of all aspects of biological molecules, biochemical processes and laboratory procedures in the field.

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pET28a(+) frustrations

Postby RichardvanLeeuwen » Fri Apr 01, 2011 12:37 pm

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? :(
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Postby patlangic » Thu Apr 07, 2011 5:36 pm

Are you sure that the Shrimp Alkaline Phosphatase you used is really working? And how did you recover (or purify) your plasmid after dephosphorylation? Have you tried ethanol precipitation or something? I also use pET28a(+) as the expression vector but dephosphorylate it by rAPid alkaline phosphatase kit (Roche), which needs no recovery step. I am not sure the same holds for Shrimp Alkaline Phosphatase, maybe you could check for it..
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