More Molecular Biology Weirdness
I keep having odd luck with gels here. My latest escapades involve screening reactions. This is where bacteria containing a plasmid with the gene of interest inserted into it are dropped into a PCR machine, and one can find which clones have which inserts. Because the insertion is not a perfect process, several colonies will have the plasmid but not the insert. We add a chemical to the growth medium that turns blue when digested, and the insert disrupts a gene (not normally present in the bacteria) that enables the bacteria to digest the chemical, so those colonies that turn blue have the plasmid but not the insert, and those that stay white have both. (All bacteria have at least the plasmid, though, because the plasmid contains antibiotic resistance genes as well, and we add antibiotics to the medium too, which kills off (or at least stops the growth of) bacteria that do not have the plasmid.)
Clear enough? Well, so I have colonies, which are tiny, and as far as I can tell, white. I set them up in the screening reaction but also grow them on a fresh plate, so that I will have more colonies if I need them. Normally, the blue-turning chemical is not added to that new plate, but I have been adding it anyway, and in this case, I found several of my colonies a nice blue colour. I screened them all anyway, and a good thing, too, because the blue colonies have the insert, and the white ones do not! If you have followed along with all of this, you will see that this does not make sense. I cannot explain it. I am going to grow them all up in liquid culture anyway, because the screening PCR should be more reliable than the colour-changing (which can be subjective).
That was my first gel of the day. My second gel, of another screening reaction, turned out perfect. Even the blue and white matched up. I am very happy. I want to grow these cultures up in liquid media as well, but I will have to wait until after I get back from Buffalo, as they will need to be tended the next day. Interrupting the lab excitement is not fun, but on the other hand, I have something to look forward to upon my return!
And meanwhile, my labwork is done for the day. There is nothing left to distract me from my reading! Nothing, that is, except for cleaning and tidying and packing for the trip....
Clear enough? Well, so I have colonies, which are tiny, and as far as I can tell, white. I set them up in the screening reaction but also grow them on a fresh plate, so that I will have more colonies if I need them. Normally, the blue-turning chemical is not added to that new plate, but I have been adding it anyway, and in this case, I found several of my colonies a nice blue colour. I screened them all anyway, and a good thing, too, because the blue colonies have the insert, and the white ones do not! If you have followed along with all of this, you will see that this does not make sense. I cannot explain it. I am going to grow them all up in liquid culture anyway, because the screening PCR should be more reliable than the colour-changing (which can be subjective).
That was my first gel of the day. My second gel, of another screening reaction, turned out perfect. Even the blue and white matched up. I am very happy. I want to grow these cultures up in liquid media as well, but I will have to wait until after I get back from Buffalo, as they will need to be tended the next day. Interrupting the lab excitement is not fun, but on the other hand, I have something to look forward to upon my return!
And meanwhile, my labwork is done for the day. There is nothing left to distract me from my reading! Nothing, that is, except for cleaning and tidying and packing for the trip....