4. Agrobacterium

After confirming your sequence is what you believe it is, you're ready to move to Agro. There are lots of details about selecting Agrobacterium that works for you, but let's dig into the transformation first.

Agro Electroporation:

So, in our lab, we usually electroporate our Agro. The simple principle behind this is that a brief jolt of electricity shocks the membrane of the bacteria and allows for plasmids to be uptaken by them. 

So, you should have antibiotics that allow only agrobacterium to grow and also antibiotics that allows for only bacteria with your plasmid to grow.

        For example, GV3101 grows with gentamycin and rifamycin, so use this while culturing those cells along with whatever bacterial resistance gene you have in your backbone (spec or kan usually).

Once you have colonies, you should do a quick PCR screen to make sure your plasmid is 100% there. If you aren't using a recA- strain, you should also consider sequencing again by extracting plasmid and retransforming E.coli to make sure your plasmid isn't scrambled, but this is more up to your discretion and funding availabilities. 

Once you have it confirmed, you can start treating it for transformation as your instructions guide.

Side note: I would love to go into Agro diversity, but my presentation doesn't have the time, if you want this, let PlantGene know!

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