Hi,
How can insert my all sequence of my WRKY TFs in vector- E.coli:
I want start to study the function of my WRKY gene to abiotic stress (nitrogen) but I have problem because I want insert all sequence of my gene to know what is the function of this gene by incresing the levels of stress.
Anyone can help me in find the way to do that, any advice i will be happy for it.
Thanks in advance
Saddam BABA
If i did understand well you want to characterize the function of a WRKY TF during N limitations, but i do not understand how you want to do this. You mentioned that you want to introduce the complete sequence in a vector, this should be easily achieved just by amplifying your TF sequence from genomic DNA and insert the PCR into a generic plasmid (pGEM, pJET).
Now if your real objective is to create transgenic lines to analyze the function of your TF, then you could use gateway technology to achieve this objective. Briefly you need to amplify your complete TF sequence by PCR, insert the sequence in a donor vector performing a Bp clonase reaction (usually pDONR 221), next you need to recombine the sequence to a destination vector performing a LR clonase reaction and finally use your working transformation method.
Nice and clear explanation by Lenin Yong and Stefano D'Aessandro
Many thanks
Saddam BABA
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