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.

Categories: Ph D posts

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ABugsLife

A Ph. D graduate in Microbiology, residing in Victoria, Australia. Currently working in multiple locations but still in the STEM field. 👀 🦠 🧫 🧬