First week of benchwork complete!
I have now had a week to do some work in the lab. I pushed hard to get some samples ready to facilitate the sequencing lab for our undergraduate genetics class. On Monday and Tuesday, I extracted DNA from 74 of the individuals. Wednesday, I set up a PCR (a technique used to make lots of copies of a single region of DNA) to amplify the same gene I used in the original study. Thursday, I ran a gel to make sure things worked and purified the DNA, which I sent out, together with the samples from our genetics class, for sequencing. The company that does the sequencing works crazy hours (which is part of why I use them); UPS delivered my samples to them at 3 am (according to the email update I received) and my sequence files were in my inbox by 7 am! I got sequences back for 59 individuals and already there is interesting news!
It was really hard to pick which samples to do first, I chose to go with one new site from Minnesota, the new site from Iowa, the site from Ohio, and the site from southern Illinois. While the sites from Minnesota and Iowa had no genetic diversity at this one gene (each population had a single DNA sequence), the single sequence in those populations was identical to those in Wisconsin (across the Mississippi River) and not to eastern Missouri (on the same side of the Mississippi). Given that there is evidence of the Hudson River blocking the movement of Nigronia, I didn’t expect to see signs of crossing the Mississippi. The site in Ohio had a lot of genetic diversity and both families of sequences that I found in the original study were still present, so that’s a really good sign.
The paragraphs above make science sound like it is on the television: fast and simple. Just to give you a better sense of what’s going on, tomorrow I’ll be starting extractions for 176 individuals. The first part of the process is labeling tubes. For each individual, I need three tubes plus a spin column. Today, “all” I and a friend did was label tubes. Five hundred twenty eight tubes, to be exact (the picture at the top of this update). That was all we did for six hours (it’s now five hours after I left the lab and my thumb is still numb). Two thirds of the tubes need information on the lids and on the sides as a backup. Now that the tubes are ready, I can do the next step: measuring head widths and isolating tissue. For each insect, I am using digital calipers to measure the head width 2-3 times to get a repeatable width. Then I remove the head and digestive tract (so we don’t get contamination from their prey), so I can take the first two thoracic segments (the ones with the legs) for the extraction. I put the parts of the body I’m not using into a tube for long-term storage (in case we need to extraction DNA again, or a student wants to study gut contents, or someone needs to verify the identification of the species) and put the thorax into a working tube for the extraction. If you want to see more about DNA extraction, here’s a link to a video I made for my population genetics class during the pandemic: https://youtube.com/playlist?list=PLxzjqM5lIz-5SQh10DVy28AXdEaw66nLF
Getting to the next round of sequences will be slower because I’ll be using a different technique to get there, but I’ll be able to get a lot more sequences all at once (the plates that the first batch went out on can only handle 94 sequences at a time). I’ll let you know what I find out.
Thursday I’ll be hitting the last field site, putting a close to stream visits for a while.
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