Sorry forgot to add that agro inserts into the genome randomly and can do so multiple times. The binary vector has a t-dna region delineated with a left and right border motif. You will see articles citing circuits that read like LB-MCS-Pnos-kanR-Tnos-RB. Your gene of interest along with appropriate promoter and terminator goes in the mcs like any ecoli plasmid. The tdna region always has those borders and a preassembled circuit for expressing a selection marker typically an herbicide resistance gene. That way you can kill off any non-transformed tissue and regenerate from only the transformed cells. If you are doing a transformation experiment you may want to regenerate a few plants to account for multiple insertions which may or may not get silence in the next generation. Multiple inserts can be considered as viral load and it will get cut out altogether. To check for insert count, grow transgenic plant until it makes seeds, then plate seeds in vitro onto media containing the selection herbicide and allow to germinate. You'd want around 50 seeds Per plate.
Once the seeds sprout, you'll notice some didnt germinate, some are happy and green, and if you are lucky, some are pale or white. If all germinated seeds are green, you have two or more inserts. You basically want a mendelian segregation of 75% resistant and 25% susceptible. At that point you know its just one insert.
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I'm currently an undergrad studying molecular biology, and I recently joined a community lab after taking biochemistry lab at my school. In my class, we transformed a colony of E. coli using egfp and I thought it was really cool, and I've kind of been obsessed with trying it with other organisms as well, so I wanted to see if maybe I could do the same thing with a plant. After spending a few days at the library and searching through a couple of scientific journals, I found that I could do a similar procedure with agrobacteria, which can use a disarmed tumor-inducing plasmid to inject a segment of tDNA from another binary plasmid into a plant genome (I'm still not 100% sure how that works or where in the genome it gets incorporated, but I know it works at least). I would like to try and put egfp into a plant and have it expressed at detectable levels. I've had people tell me it's not too difficult to do. If any of you have done it before, or if you know what is involved, could you possibly give me some pointers on how/where I can order the appropriate plasmids, and which strain of agrobacteria I can use? I found a couple of protocols, so I have a general idea of what I'm gonna do (floral dip), but I'm still a bit iffy on how to select the right plasmids and bacteria, and where exactly on the binary plasmid I should ligate my gene of interest. Any help is much appreciated :).--
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