Part 2 of the clone woes (as I’ve dubbed it) is the current update on what’s happening.
Just as a reminder from part 1, I’m currently trying to clone three different plasmids.
Due to some technical issues involving the restriction enzyme EcoRI, I’ve had to revise my strategy.
I got the red insert from plasmid #4 into plasmid #2 already (let’s call this new plasmid #8). I transformed this new plasmid into my Coxiella strains last week, so I’m currently waiting for my picked/isolated colonies to grow up. Once grown, I can proceed to step 3 of my Coxiella transformation steps (see previous post) to check whether the protein that plasmid #8 is instructing Coxiella to make is being produced (let’s call it the yellow-red protein).
I’m trying to get the green insert into plasmid #1 at the moment. Because I’m using one enzyme to cut, it means the insert could go in backwards (yes, there’s a direction to these things! With the way it’s drawn above – which is quite standard – the direction is clockwise). I’ll check to make sure I only pick the ones that have been inserted in the correct orientation.
I think I have plasmid #9 as well. I’ve sent it off to get it sequenced to make sure there aren’t any errors. Fingers crossed, but this means I’ll try to put this plasmid into Coxiella soon as well.