What's the point (mutation)?
This story begins with a bit of a preamble. My awesome boss at my current job said it was OK for me to make some protein constructs for my PhD, despite the fact I won't be working for him in a few week's time and that it will be costing the company money rather than coming from my stipend. He said 'so long as you get some template DNA you can make some constructs'.
I wrote to the PI of the lab I will be working in, who obviously jumped at the chance of free construct design and so sent me some template DNA. Me being me, I gratefully received the plasmid and instantly transformed it for miniprepping two days later.
Roll forward two days. Three clones have been miniprepped and sent for sequencing. Data comes back from this and I happened to drop it into ClustalW to check the alignment...then disaster strikes. I realise that all three have a P48A mutation in them. Hmmmm, strange. This obviously couldn't be down to chance, one I could understand but all three?!
I wrote back to the PI and mentioned this to her. A very quick reply popped up in my inbox.....somebody has mislabelled tubes and sent a mutant copy rather than the WT. Eep. Her reply said 'do you want me to send you some of the WT plasmid?'
Now, having lost a week making DNA, sending stuff for sequencing, I thought 'you know what? I'll just use this as a template to mutate the DNA back to the original, how hard can it be?'. Oh god, was I wrong.
Primers ordered, I tried three different ways to mutate this template back and frustratingly none of them worked. Different annealing temperatures, different elongation times, different gel purification protocol. Darn it, nothing was working and here I was running out of time.
With my tail between my legs I emailed my PI back and asked her in a pleading voice (if that is even possible in email) if she could send me some WT plasmid. Meanwhile, not really trusting that this would even be the correct sequence for the plasmid, I decided in a fit of panic to order myself a synthetic gene with the correct sequence but also to codon optimise it. That way, I knew the primers I had previously ordered would bind, the sequence was correct AND that it should express ok in E. coli.
The wait was on, I had three weeks left before I was due to leave the company. WT plasmid was in the post, synthetic gene was being synthesised and couple this with the other work I had to do, my stress levels were through the roof.
Pretty good going seeing as I haven't even started my PhD yet!
