Gustafson_Picture25, a photo by REU SLAWR on Flickr. Setting up DNA Testing Samples.
By Ryan Gustafson:
The main goal for the week was to obtain snails for the DNA test. We are half way through the program at this point and still hadn’t had the chance to do any tests on the thing that we are actually studying. Considering this, Tony put extra pressure on Libby to contact the person that agreed to provide us with snail samples. Libby and Tony both called and texted the person and eventually he got back to them but didn’t seem to be very happy about the situation, so we began to more seriously consider our alternative. The alternative was to collect snails on the north side of the lake, something that still required permission from the Fish and Wildlife Service. Libby had been calling the Fish and Wildlife Service for a couple of weeks and finally heard back from them on Monday. The response was not exactly what we wanted because the person just said that they think it should be fine, but he was not 100% sure and said that he would have to ask another person who was out of the office for the day. Later on Monday we ended up getting a sample consisting of one snail from the person sampling on the reservation, which indicated to us that we really needed to find them elsewhere or we would only end up with a very small sample. By Wednesday at noon we had not heard back from the Fish and Wildlife Service and with time running out we decided to sample on the north side of the lake anyway. The impression that we got from the person that we had talked to from the Fish and Wildlife Service on Monday was that there would most likely be no problem with us sampling small invertebrates, so we decided to go through with it.
Snail sampling began at the north side of the lake in the town of Lakeside. Libby, Cheyenne, and I began by looking at the shallows of some of the docks and public swimming areas. The water at the docks was a little deeper than would be ideal for snail sampling and from above we couldn’t see anything that looked like snails. The docks also did not have much aquatic plants and so they didn’t seem like a prime habitat for snails. The public swimming areas were dominated by coarse sand and gravel and so while the water was shallower, it did not allow for the growth of the aquatic plants that provide snails with habitat. One of the swimming areas that we investigated was near an area that had thick aquatic vegetation, but it was mostly cattails rather than the flowering rush. Even though there was no flowering rush, we decided to suit-up and investigate more closely. I put on the chest-high waders and a pair of boots, while the girls put on thigh-high wader boots. Cheyenne went in first but it turns out that the mud at the bottom was pretty thick and so her boots were not tall enough to allow her to be in the water. After Cheyenne got out, I got in with no problems, using my chest-high waders, and began my hunt for snails. Each step that I took kicked up significant amounts of mud that would dirty the water before I could get a good look for snails. I tried looking on the bottoms of logs and on the various aquatic plant stems but could not find a single snail. After about forty minutes, we decided to give up on this location and to drive to Wayfarer State Park where we heard we might be more likely to find snails.
On the drive to Wayfarer State Park, we decided to pull off at Fennen Slough fishing area, which turned out to be just what we were looking for. After looking around a couple of different areas at Fennen, I found a spot that offered up a few snails in just a few minutes. I eventually let Cheyenne and Libby know about my findings so they put on their boots and joined me. They brought jars with them for me to put the snails in and as I began stepping around in front of them they noticed that more snails came up whenever I took a step. This finding allowed us to collect roughly 150 snails in about twenty or thirty minutes. We each put about fifty snails in our jars, which we filled with water and sealed up for the ride home. On the way back we grabbed some huckleberry shakes and buffalo burgers to reward ourselves for a hard and hot day’s work. Once we got back to the lab, we placed two of the jars in front of a lamp to coax the parasites out of the snails and into the water, and the third jar out of the light.
On Thursday morning we arrived back at the lab to try to get some DNA from the snails, which would hopefully contain some parasite DNA. The first thing that we discovered when we got to the lab was that all of the snails had died overnight. Our suspicion was that we did not give them enough air, maybe that they were too crowded or did not have enough food. Even though the snails and perhaps the parasites were dead, we still tried to get DNA from them. We used the DNeasy Plant Kit for getting DNA from plants but just applied it to our snail samples with a few modifications. The DNA extraction process was a very long one that started around 11:00 AM and didn’t finish until 5:00 PM. The DNA extraction process allowed us to learn new techniques and improve the standard techniques that we had already learned. Libby also explained to us the purpose of each chemical or step in the process, which was extremely helpful and I am extremely grateful to have had someone to facilitate our learning while we were using the kit. After the many hours of work put into the process, the results were not what were expected. The machine and computer program that we used to determine the presence of DNA did not provide results that indicated any significant amount of DNA in the samples. We plan to check our samples by doing DNA gel electrophoresis next week, which should indicate whether any DNA is present in the samples. If there is no DNA in the samples then it could be that we should have used the DNeasy Blood and Tissue Kit instead of the Plant Kit. Libby assumed that they would not be much different, but perhaps they are more different than she expected, so we will also try the Blood and Tissue Kit next week.
During the fifth week of the REU, significant progress was made on my project by finally collecting snails and attempting to extract DNA from them. While the process did not go as expected, we are going to check our results on a gel next week and possibly use the Blood and Tissue Kit. We may also sample more snails next week, being sure to give them enough air, food, and space. I will also complete the finishing touches on the introduction which I have also improved and added to over the last week.