Some Weird Stuff with Wasp DNA, and CAKE!!!

Today was a busy day. So busy, in fact, that all the work could not be contained within one building. Also, I didn't even have time to do any culturing (fortunately, the army of undergrad workers was there to help out, but tomorrow, I won't have that luxury).

So, as the title of this post suggests, we did some advanced stuff with wasp DNA that was entirely new to me. Using the QIAquick brand kits, we did a gel extraction of wasp DNA, and then messed around with it in a NanoDrop machine to determine the concentration of DNA within, in nanograms per microliter. But why did we have to jump through all of these hoops to get usable DNA when we already started with usable DNA, and had a lot of it? The answer: preparation for sequencing. Apparently, there's some sort of controversy or uncertainty with the DNA of the various Encarsia inaron wasps, and so we're painstakingly working towards sequencing the DNA of the Italian population of E. inaron and 8-times intergressed local E. inaron. If I remember correctly, we did a series of chelex DNA extractions a while back, and used those samples today.

First, we mixed all of the similar samples (same DNA, same primer pair) into their own large samples, and ran those for about 40 minutes on a small agarose gel. Once that was completed, we took the gel over to the neighboring Riehle (pronounced "really") Lab and cut out the DNA bands. To see the DNA bands, the we placed the gel on a UV light source. Since in this gel we used SYBR safe instead of SYBR green, the bands were nice and uniform, instead of "all over the place." After cutting out the bands, we had to add a series of buffers to their tubes, and incubate them at 50 degrees Celsius until the mixture all melted together, at which point a process similar to the kit extraction took place (for more info on that, refer to a previous post, or remember that stuff we did earlier this year with E. coli, those of you who were in Capstone Biochemistry).

As I mentioned, we had to go to another lab/building in order to figure out how concentrated our DNA product was, so we headed over to Life Sciences South and used their NanoDrop machine. It's quite a cool piece of technology! All we had to do was put a 1-microliter drop of our DNA product onto the sensor, and within 30 seconds, the machine told us what the concentration of DNA was. It determines this by comparing its absorbency to the absorbency of the solvent in which it is dissolved (in this case, PCR water). We needed to know the concentration in order to determine the extent to which our DNA needs to be diluted before it is ready to be sequenced. Yes, certain lengths of DNA need to be at a certain concentration in order to be properly and precisely sequenced. The formula is quite simple, all we needed to do was multiply the length (in base pairs) of the region of DNA specified by our primers by 0.02. The resulting number is the concentration (in nanograms per microliter) that the DNA must be at. Of course, determining the length of the DNA fragments is not always easy. Although some of this information is lying around in the lab, and some of it is available on the internet, some isn't, and so we had to make educated guesses. We used 5 types of primers: 28S, CO1, EF, gyrB, and Cardinium. They all have more specific names, but those are the general families of primers. I know that 28S is a eukaryotic gene, and EF is made from Encarsia pergandiella, and "Cardinium" refers to a gene in the bacterial symbiont, Cardinium.

Oh yeah, we're having cake on Thursday. I'm hoping for red velvet!!!