Posted in Ph D posts

The clone woes

In my case it was just human error.

This piece is filled with cloning jargon! You have been warned!

In a previous post, I explained my upcoming experiments in a bit of detail. Prepare for an update on how that’s all going!

FYI highly recommend reading the previous post to make sense of this current one.

I’m currently wandering around the cloning stage of my upcoming experiment.

I successfully managed to generate my first plasmid DNA (‘Plasmid #2’), so I basically completed step 1-10 already.

Each little pair of scissors denotes a restriction enzyme, which can cut very specific sequences of DNA. You can see how many different combos I’m using throughout.

But now that I have Plasmid #2, I now need to make the rest of the plasmids I actually need for my experiments. Enter Plasmid #3, #4 and #5…

Basically I have to put a green bit, a red bit, and a dark blue bit, into Plasmid #2. In the end I want three, brand new plasmids, each with different inserts.

I was basically trying to cut my inserts out of Plasmids #3, #4, and #5, then put it into Plasmid #2.

But even with the odds being somewhat against me, this process just wasn’t going as well as it usually does. Something was wrong.

And it dawned on me.

Note the red arrows. The problem was EcoRI.

I completely forgot that Plasmid #2 already had an EcoRI site (which you can see next to the pink bit on my diagram). So what was happening was, when I cut with EcoRI, I lost the pink and orange bits- both very important components I definitely can’t do without!!

So we’re going to revise our methodology to accommodate this issue. Glad I remembered now rather than later, but… *sighs*

And so the cloning continues…

Author:

A wet-lab based Bacteriology Ph. D student residing in Australia.

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