I want to do plant genetic transformation with my gene of interest. For that reason, I need to construct the vector Ti plasmid so that can go for Agrobacterium-mediated plant genetic transformation. How I will cut the plasmid, what will be the enzyme and how to make it ligate? Anybody who has already done such work can help me by providing me the protocol?
Thank you in advance.
Hi Shahina,
The first you need to know is which enzymes are at your gene of interest/Ti plasmid. Use a program like Snapgene to see with restriction enzymes are compatible with Ti plasmid vector bonding site. If there is compatibility, its better to use 2 different enzymes for each extreme, so you don't have a problem with the direction of our insert (like if you use 1 enzyme, you can introduce your insert in the opposite direction). If you don't mind the direction, go ahead. Then for each enzyme there is a compatible buffer, check it! For NEB/Promega you can enter to the website and find the buffer compatibility tables that will tell you which to use according to your combination of enzymes.
You can make many protocols, and probably you will need to optimize them. But in general I can give you this order:
1-Extract DNA: commercial kits can work well.
2-Cut your DNA or make PCR to extract your fragment: if you don't find any enzyme compatible, maybe you need to considered to add some adaptors in the end of each extreme of your gene of interest and the cut them with the enzymes you want.
3-Cut your Ti plasmid.
4-Run both o agarose gel: I used TAE gel and choose the % of gel according to your fragments size. EVALUATE YOUR RESULTS: here is a QC stop to see if you obtain what you expect. If the fragments are on the size that you plan, continue. If not, considered that or the enzymes don't work or something went wrong.
5-Cut your desired fragments from your gel and clean them. You can use Promega kits to clean the gels. To see the fragments, I use Ethidium bromide and UV lamp.
6-Ligate the fragments: use Ligase enzyme according to manufacture's protocol.
7-Clean your construct. Use again a kit like Promega or other compatible.
8-Sequence your vector! You almost there! Just check your insert is there, using primers from the Ti plasmid to the insert (so you can know if is really there).
9-Done. Now choose how you are going to make the transformation. I used to work with electroporation.
Hope this help you.
All the best!
Laureana
Shahina Akter You may use the following one:
Article Construction of Disarmed Ti Plasmids Transferable between Es...
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