You know the feeling. You’ve attempted some cloning for the first time in months. The PCR product (all bloody 3kb of it) has come up fine. The clean up has been successful (I haven’t lost my product!! Yey!!) You’ve stuck a thymine on the end, ligated it into a vector, heat shocked the cells… and now it’s 9am and you’re looking to see what’s grown.
I have colonies!!! Huzzah!!!
…. Oh wait …. they’re blue. Bugger.
Quick microbiology lesson for the uninitiated:
To put a gene in a bacterium, you first put it in a piece of circular DNA called a plasmid. Bacteria use these to swap interesting genes like genes for antibiotic resistance, so they’re used to picking up DNA in this form.
The plasmid also has some other genes on it. One of them is called lacZ, and it allows the bacteria to use a chemical I spread on the plates called X-gal. When the bacteria use this gene they produce a by-product that is bright blue.
If my gene is correctly inserted then it will sit in the middle of this lacZ gene, stopping it from working. So white colonies contain the plasmid with my gene in, and blue colonies contain the plasmid without my gene in.
Time to start over…