I woke up at 7am this (Saturday) morning to an amazing realisation: I’ve wasted weeks of work because one of my tests was completely futile.
I’ll tell you the steps of my work; see if you can pick where I went wrong.
Aim: Create a copy of an E. coli gene on a plasmid.
- Amplify tiny piece of DNA (gene) from E. coli, using PCR.
- Put gene inside bigger piece of DNA (plasmid).
- Put plasmid inside of E. coli so that it multiplies and makes lots of plasmids for you.
- Check whether plasmid inside E. coli has the gene by doing a colony PCR, which contains all the DNA inside one E. coli colony.
- Regrow colonies that contain the gene, get plasmids, continue with more steps.
I’m up to step 5 at the moment and I’m having trouble regrowing the colonies, which is why I have been thinking about this. Anyone guess what’s wrong with what I did?
Well, I’ll tell you…
I’m cloning (that’s what this whole process is called, has nothing to do with creating sheep or dinosaurs or copies of humans) an E. coli gene; I tested all the DNA inside my transformed E. coli cells to see if the gene was in the cell.
Of course it’s in the sodding cell! It’s even the same strain of E. coli that I got the gene from in Step 1! There’s no way to tell with PCR where you’re amplifying DNA from: it could be from the genome or a plasmid. So my results told me that all the colonies I picked had the gene, presumably on the plasmid. Since I haven’t been able to grow them again, I don’t think they do have plasmids, or they lost them. If you have any knowledge of what I’m talking about you’ll know that we use antibiotics to make sure there’s a plasmid — I did, but maybe over time the antibiotic broke down or something.
I feel stupid! Not as stupid as I could feel though, because my lab supervisor told me to do what I did. Normally the technique would be okay because he works with genes from a different species, which would not be in E. coli… I think I can rectify the situation without too much extra work, but I’ve been battling with these cells for at least two weeks now. What a waste of time!