Month: May 2007

Argh! Blogstipated again!

I actually am still here, but Spring 2007 finals went straight into Summer 2007 classes, and it’s pretty intense. A six-week Immunology and Immunology lab alongside an entire semester of “Speech” class crammed into 4 weeks means I’m pretty frantic at the moment.

Although the semester is over and the paper turned in, I am still working on my 8 beer-bacteria isolates. In fact, re-doing “colony” PCR with a few µl of broth culture seems to have actually worked pretty well:

To the left, there, you can see the results. Interestingly, if you look just to the right of where the “wells” are in the gel, in several lanes you can make out a visible band of obviously large DNA molecules, which I presume are genomic DNA from the bacteria. The fact that they seem brighter in the lanes where the 16s band at the end (or at least, I’m ASSUMING that’s what that is, and my reaction worked) is dimmer tends to support my suspicion that I’m “swamping” my reaction with the template DNA and making it hard for the reaction to work sometimes.

Anyway, once I’ve worked out how to actually do a useful Restriction Enzyme digest to get an idea of how many of my isolates are really unique, I should hopefully be about ready to get them sequenced so that I can identify them. Then, I’ll be ready to try them out on my thesis project. Meanwhile, both my current DNA extraction from my recent thesis project sample and the amplified 16s gene bands for my lactic-acid bacteria from the beer are sitting in the freezer.

I’ve got an intense couple of days now, but hopefully I’ll have time to blog a bit more over the weekend.

Colony PCR – because DNA extraction protocols suck.

If you’ve got a culture of a single type of bacteria and you want to identify it, the standard method is to figure out the sequence of one particular gene, the 16s rDNA gene. That is – it’s the gene which encodes the a piece of RNA that gets used by the ribosome in part of the process of “reading” which amino acids to link together to make a particular protein. This is something that every prokaryote known has, and parts of it are conserved, so they’re similar enough to compare, while other parts can vary a lot, providing enough “difference” to tell different organisms apart.

To figure out the sequence, you use PCR to “amplify” this particular gene, making lots of copies of it so that the sequencing machine can clearly see the signal from each part of the sequence. And before you can do that, you have to get the DNA out of the cell relatively intact.

That part can be a pain. There are lots of different ways people have come up with (and made special canned “kits” out of) – you can use chemicals to try to dissolve the cells and let all their guts (including the DNA) out, you can try to mash them up with tiny glass beads in a “bead-beating” machine, you can stick them in a blender, you can even just boil them for a while…then usually you go through several steps of centrifuging and mixing with different chemicals and then centrifuging again until you’ve hopefully finally got the DNA out and gotten rid of most of the other cell bits. And, hopefully, you haven’t accidentally chopped up the DNA too much to use in the process.

Fortunately, there’s a trick you can sometimes use, referred to as “Colony PCR”. In it, you literally just touch the top of your colony of cells and shake them off directly into the PCR tube. Then you just include an extra 5-10 minutes of 95°C heating to hopefully cook open enough of the cells to release DNA (and cook the cell’s enzymes to death so they don’t degrade the DNA and interfere with the PCR).

Not real reliable if you’re trying to do anything quantitative, like trying to figure out how many copies of a gene are in each cell, or trying to get an accurate estimate of how many cells of one type or another are in a mixed culture, but if you just need as much of a particular bit of DNA as you can get – such as for sequencing – a lot of people use this.

I just tried it on my Lambic isolates. Two of the 8 bacterial cultures worked beautifully. I’m pretty sure the problem with the other 6 was just the sheer amount of bacteria I ended up adding to the reaction – too much seems to “swamp” the PCR process and keep it from working. I’ll try it again this week. But it does seem to indicate that it works, at least.