Hello Everyone!
I am new to plant molecular biology and i have a doubt. Is it always necessary to clone a plant gene in an entry vector (such as pJET, pGEM®-T Easy vector) before cloning into a destination expression vectors such as pCAMBIA? what are the advantages? Thanks in advance.
Hello Hariharan Balasubramaniam ,
it is not necessary to clone a plant gene in an entry vector. However, if you want to use your gene with another promotor or antibiotique or a gfp tag, it's easier to already have it an entry vector. Usually recombination between entry vector and destination vector are done with specific enzyme and it's really easy.
1. You stick your gene into those vectors (ex. pGEM-TTM Easy) first, so you can multiply them (plasmid with backbone and your gene) and get enough plasmid material for later experiments. You can also sequence your gene to check whether any mutations occur before you subclone your gene into Agrobacterium vectors. pGEM-T Easy vector can be used to sequence your gene after you ligate your gene (a PCR product) into it.
2. I would not specifically call plasmids such as pGEM-T Easy vector a 'entry vector'. When you mentioned 'entry' and 'destination' vectors in the question title, it gave me the impression that the question is something to do with the GatewayTM cloning system, which use such terms (see attachment).
Dear Dr. Yuan-Yeu Yau, Thanks again for providing me a clear overview on 'entry' and 'destination' vectors.
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