Hi
While using the MCS (Polylinker) of the pCAMBIA vectors, whether, it is necessary to clone the insert of interest along with the promoter and terminator or only the insert of interest is enough?
I think, since the polylinker is not possessing the expression cassette, the insert of interest must be cloned along with the regulatory sequences like promoter and terminator.
Please educate me in this regard.
Thanks in anticipation
BASAVARAJ
Yes. The MCS region of pCAMBIA vectors does'nt have the promoter and terminator. Alternatively, for example if you opt to use pCAMBIA1305.1, you replace the reporter gene (GUS) and instead clone your gene of interest utilizing the available restriction sites viz., NcoI, SpeI on one side and NheI, PmlI on the other side. By this way you don't have to clone the promoter and terminator additionally.
If its pCAMBIA 1302, use any of these sites NcoI, BglII, SpeI, PmlI, Bst EII, That will place your target gene between 35S promoter and nos terminator; Selection of these sites will also decide if GFP will be fused to your target or not.
Thank you all for the kind and prompt reply.
However, my doubt was, if the MCS has to be used, whether the regulatory sequences (Promotor and terminator) are to be cloned compulsorily along with the gene of interest . Because, there must be an eukaryotic expression cassette to express any gene in the plants, which is not there in the MCS.
Sincerely
BASAVARAJ
Yes, in that case you must include an intact gene expression cassette comprising the promoter ---- gene ---- terminator
If you do not make your gene cassette complete (Promoter-Coding seq-Terminator) your gene may not express well.
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Molecular Methods
- Cloning