CAMBIA Vector Cloning Strategy and Trouble Shooting

A few points about the pCAMBIA cloning strategy:

The pUC18 polylinker was used in some vectors, but pUC8 and pUC9 polylinkers were also used to simplify the choice of cloning enzyme. In the age of PCR, it is no longer necessary to have a large number of cloning sites. The smaller polylinkers also eliminate potential conflicts from sites such as SphI (which has an ATG) or XbaI (which has a TAG). This makes other sites in the vector more useful (such as the SphI site outside the right T-DNA Border, or the SacII site outside the left T-DNA Border).

Plant selection genes in the pCAMBIA vectors are driven by a double-enhancer version of the CaMV35S promoter and terminated by the CaMV35S polyA signal. NOTE that this 35S promoter can have an enhancer effect on the expression of other genes in the same cassette, so gene expression results using pCAMBIA derivatives in which portions of this promoter are still present should be interpreted with caution. Furthermore, it is your responsibility to check whether the 35S promoter or any other components you use are subject to patents in your country. You can find help with this at CAMBIA's Patent Lens website.

Reporter genes feature a hexa-Histidine tag at the C-terminus to enable simple purification on immobilised metal affinity chromatography resins. The sequence for this tag occurs between the first NheI site (there is a second NheI site in the pVS1-rep that we didn't eliminate) and the unique PmlI site. Genes of interest may be inserted in place of the reporter gene. Insertion without a stop codon and in frame at the (first) NheI site will append a hexa-Histidine tag to your protein of interest. Insertion without a stop codon and in frame at the PmlI site will append a stop codon. Insertion at the BstEII site will add neither a tag nor a stop codon (so you may want to ensure that a sequence inserted here contains a stop codon).

tDNA sequence of pCAMBIA Vectors

The tDNA sequence for pCAMBIA vectors can be downloaded by clicking TDNA v310 (info)

What concentration of antibiotics should I use?

pCAMBIAx1xx vectors with spectinomycin/streptomycin resistance (aadA/aminoglycoside-3??-adenylyltransferase) in bacteria: use 50-100 µg/mL in E.coli & A.tumefaciens

pCAMBIAx2xx vectors with chloramphenicol resistance (cat ? chloramphenicol acetyltransferase from E.coli Tn-cam204) in bacteria: use 10-25 µg/mL in E.coli & A.tumefaciens.

pCAMBIAx3xx vectors with kanamycin resistance (aphIII/nptIII/3?5??-aminoglycoside phosphotransferase typeIII from Enterococcus faecalis pJH1) in bacteria: use 50µg/mL in E.coli & A.tumefaciens.

What concentration of plant selection antibiotic should I use?

CAMBIA Login