TA cloning and C-terminal tag - does this work!!! - (Apr/05/2010 )
I was just discussing an issue about TA cloning with a colleague and wanted to get more opinions on this!
So, the motivation: in vivo expression of a protein with a C-terminal tag by generating stable transgenic lines (in my/our case in plants).
The method: Cloning the gene of interest with removed stop codon into a gateway compatible entry vector and from there into the actual gateway vector containing the desired tag.
And the problem: Both of us used TA cloning for the entry vector and in our discussion we realized that this should theoretically result in a frame shift at the 3' end of our gene of interest because of the T/A overhangs. And eventually this means that also the tag is not properly expressed!
Is this right!?
All the best
stupidity won again - problem solved!!!