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The clone woes (part 2)

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.

The original plan.

Due to some technical issues involving the restriction enzyme EcoRI, I’ve had to revise my strategy.

The problem (red arrows) – I lost the red and yellow bits when I cut with EcoRI.
You can actually see the (really) faint band between 1-1.5 kb that indicates the pink and yellow portion was falling off. It only happens when I have EcoRI in the mix. Also FYI- this is what a DNA gel print out looks like. This was images using UV rather than Blue Light.
The revised approach.

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).

Current status of the clone woes.

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 former wet-lab based Bacteriology Ph. D student residing in Australia. Now working part time at a secret location as a Communications and Data Officer. 👀 🦠 🧫 🧬

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