Thursday, July 15, 2010
This post is about work, so be forewarned...
Since I work with mosquitoes, which are usually quite abundant when we collect them, I process lots of samples at one time. After they are ID'd, each bug is ground up in a bit of liquid, and I use a robot to extract the DNA of 96 individuals at a time. Once we have the DNA, we can run various assays that tell us about what genetic markers they have. My job is to compare populations of mosquitoes for these markers and try to get taxonomic information about them.
I've been working on getting more markers. We had 8, which isn't too bad for this kind of thing, but 2 of them were kind of wonky. I was able to develop 11 more, and I expect about 6-8 of those to be suitable for what we do. To save on materials, we group the markers into something called multiplexes. I have two multiplexes, with 8 and 9 markers each.
The markers work by using primers, little stretches of the A's, T's C's and G's that make up DNA. These have particular sequences that correspond to a spot on the mosquitoes' DNA, and we then amplify enough copies of this stretch of DNA to be able to see it using a DNA sequencer that is able to tell us how big the fragment is.
For whatever reason, (gremlins, chemistry, something) one has to vary how much of each primer that is used in the multiplex. Getting that right takes a while - weeks, usually. But when it's done, each marker in the multiplex gives its signal at roughly the same strength on the DNA sequencer. It should look like the bottom panel of this picture I found on the web.
During this process, the primers (two for each marker, because the DNA is double-stranded) are in little capped tubes, and getting one assay together means lots of uncapping and capping little tubes that hold each primer. When it finally works, one can make Master Mix.
Master Mix is lots of 96-well plates' worth of reactions made at one time and frozen, so then when the assay needs to be run, I can just thaw a couple of tubes and do it, as opposed to measuring out each primer. It cuts the time down by 80-90%, easily.
The point of this post that is longer than I wanted it to be is that I have gotten to the point where I made Master Mix for one of the two multiplexes yesterday, and will hopefully make MM for the other multiplex today. It's the culmination of a few months of work, so it feels good to get those tubes of stuff made and in the freezer. Yea!