Curriculum Workshop: Worked on our poster. Got some of the text and some of the photos figured out. We’ll need to figure out how to present our data.
Research: We made two sets of .75 mg/ml gels with 3, 4, 5, 6, 7, and 8 microM FITC (40,000 g/mol). We placed the gels in 6 ml of clean 1X PBS, and monitored them on the Bio-Rad every 10 min to see the diffusion over a period of 110 min. Having collected the data, we’ll work on the analysis tomorrow.
Morning: Met with Linda to discuss the expectations we need to meet over the school year. It was good to have them enumerated. Then, before lunch, we made two sets of gels, one with 40000 g/mol FITC, and one with 70000 g/mol FITC. We used the same concentrations for each set that we’d been using with the 4,000 g/mol FITC, and they were really BRIGHT. We will probably need to use much smaller concentrations to be able to read them with the Bio-Rad.
Afternoon: Spent the afternoon reading the gels in the bio rad. DEFINITELY too bright to be of much use. Tomorrow we’ll lower the concentration so that we can collect the data on the Bio-Rad. We also analyzed the data from Monday. Got nice logarithmic curves for the diffusion into the buffer and decent exponential ones for the diffusion out of the gels. The data for the diffusion into the buffer looks far more accurate, but then, we knew it would be, since we’re reading the gels through the buffer. Possibly our final results will focus only on the analysis of the buffer, but we’ll talk to Millie about this when she gets back from Ireland.
We made two sets of .75 mg/ml collagen gels infused with 15-40 microM FITC-dextran to place into clean wells of 6.0 ml 1XPBS to watch the FITC diffuse over time. We spent the afternoon collecting the data and will analyze it on Wednesday, as tomorrow we will be going on a field trip all day.
Well, it’s been a productive week. It started on Monday with our making gel sets that we thought would give us our first real data (.75 mg/ml and 1.0 mg/ml gels with FITC ranging from 100-225 microM to test on the nanodrop to see the rate of diffusion of the FITC to the buffer). HA! We got real data all right, but it showed the amount of FITC in the buffers to be DEcreasing instead of INcreasing, which defies common sense. Finally, after getting these results consistently, we concluded that we needed to change our method, which we did on Friday morning. We only did one set for each of the FITC and the carboxyfluorascein, on the grounds that if it didn’t work, why waste the materials? However, it did seem to work. We made .75 mg/ml gels with lower concentrations (15-40 microM) of dye, and placed the gels into larger wells with more buffer in the hopes of speeding up the diffusion process. We also changed our method of data capture back to the biorad. Well, we got lots of data. Furthermore, at least initially, it looks to be going in the right direction! It’ll require a lot of analysis, not to mention a lot of repetition next week, but perhaps we’ve found the method to use to actually make progress on our initial goals.
The whole thing provides an interesting perspective into the world of research. First, we knew what we should see, and we knew that we were seeing the opposite of that. Onto problem-solving: why were we seeing the opposite, how could we change our process to correct that problem, and when that didn’t work, how could we change our data collection scheme entirely in order to see what we knew MUST be what was happening. We spent a week trying to correct our method using the nanodrop, and when we could neither correct it nor explain it, we decided that the nanodrop was the wrong avenue for collecting the data. A wasted week? No, a valuable experience, and one not too different than researchers experience routinely.
Yesterday we made two sets of .75 mg/ml gels into one of which we mixed FITC ranging in concentration from 15-40 microM (by 5’s) and into the other of which we mixed carboxyfluorascein using the same range of concentration. Today we placed each gel into a well with 6 ml of 1X PBS buffer (in the past, we’d placed the gels into 1.5 ml of that buffer), and immediately started taking measurements on the biorad. We took data on both the gel (floating in the buffer) and the buffer (surrounding the gel) for each set of wells. For both sets, we’re hoping that the decrease in concentration of the diffusing species will correlate with the increase of the concentration of the species in the buffer.
We took data periodically from 9 am until 11:30 on both sets, and took one reading after lunch. We’re working on the graphs, but I only completed the concentration with respect to time graph on the FITC gels. I still need to do that relationship for the FITC buffer as well as the carboxyfluorascein gels and buffer. However, at this point it does look as though the trend in gel concentration is certainly downward over time in the gels (go figure!) and upward in the buffer. Possibly we have found the medium we can use to complete this project. Here’s what the data looks like for the buffer for one of the gels:
We also continued collecting data on the nanodrop for the .75 mg/ml and the 1.0 mg/ml gels we made early in the week. The new data is consistent with the behavior of that project all week.
Well, with essentially only two weeks to go, I realize that we’ll probably have to change our research goals. We read the data again for both the 1.0 mg/ml gels and the .75 mg/ml gels. The 1.0 mg/ml gels continued to decrease, and the .75 mg/ml gels seemed to have increased slightly, but only slightly. Yesterday we posited two other causes for our anomalous data: that the plastic was holding on to the FITC molecules and not allowing us to see the diffusion into the buffer, and that the FITC was degrading. We did two tests with just FITC in buffer to see if the FITC concentration decreased over time. It did not. So we were stuck.
So we talked to Millie, and she agreed that our data is not behaving properly. She came up with two alternative possibilities for reasons for the oddness: 1) the diffusion rate is so fast that we’re not seeing it at all except in the initial peak. 2) The FITC-dextran molecule is so large that the “kinetics are such that the diffusion is disgustingly slow.” We already ruled out the first one, on the grounds that the small peaks we see at the initial portion of our testing time is too low to represent what we expect to be the final concentration of the FITC in the buffer. We are testing the second possibility by, first, continuing to test the gel samples that we have already made, and, second, but creating a gel set with carboxyfluorascein (the dye we used when we were simply learning the techniques), which is a much smaller molecule and so should diffuse much more quickly than the FITC-dextran. If we can detect the diffusion of the carboxyf., we will be more confident that our process is valid for this particular experiment.
Furthermore, in order to facilitate continued research with the FITC-dextran, we are going to change our process, in the hopes that we can detect smaller changes in concentration than the nanodrop may be able to detect. Our change is that we are going to go back to low concentrations of FITC-dextran (and carboxyfluorascein, in that set) in order to be able to use the biorad, which is much more sensitive. To “assist” the diffusion process along, we’re going to place the gels in large wells with 3x more buffer to increase the gradient to make diffusion happen more quickly. We will read the gels themselves rather than the buffer, in the hopes that we will detect a drop in concentration of the FITC or dye. Also, we will not be moving the gels out of the buffer, so they will remain intact and readable. Because we’ll be keeping them in the buffer solution as we read them, we will be introducing some error in actual concentration of the diffusing species in the gels themselves, but we’ll worry about dealing with that later.
So today we made those new sets of gels and will start our analysis of them tomorrow.
In the afternoon we went to visit the GE Energy plant, at which they manufacture solar panels. Very interesting and inspiring. I got some ideas of things to do with my students, from some maximization problems for calculus to a real-world problem for my lower levels students (to use an electric bill to determine how many solar panels a household would need). These issues lead to a nice discussion on the need for renewable energy and its relevance to the students themselves.
Research: We worked on continuing to test our 1.0mg/ml and .75 mg/ml gels that we’d made yesterday and Monday. The FITC concentration continued to decline in the buffer. We discussed several ideas for why this is happening and what to do about it/how to verify it. One of the graduate students suggested that perhaps FITC-dextran has an affinity for plastic and so the diffusing FITC might be sticking to the wells. Beth suggested that we dump the wells and look at the empty wells under the BioRad. When we did, we saw no appreciable signs of FITC concentration. Then Julia suggested that we mix some FITC and buffer solution in glass, test that solution, and then put it in wells to see if the FITC concentration decreases over time in the wells. We shall see. We’ll meet with Millie to discuss the whole issue tomorrow.
Curriculum Workshop: Discussed poster contents. The poster should contain:
Summary of the research goal
Significance to the world (why did NSF fund this project?)
Reflection on being in lab
Reflection/observations on intersections with curriculum
Pictures of success and failure in the lab
Pictures of instruments
Methods
Materials
We also discussed each project and how that might fit on a poster. Our power point presentation is due a week from Monday (last Monday in the session), as is the poster. So this Aberdeen group needs to get its poster cooking!
Today we tested our .75 mg/ml gels and our 1.0 mg/ml gels over time. We are getting confusing data. The concentration of FITC in the buffer appears to be decreasing rather than increasing, and we are flummoxed.
We worked and worked on perfecting our technique to reduce error that way, and we rinsed our gels today to keep the transfer of extra FITC initially to a minimum, but still the data shows decrease. One question to ponder is whether it’s a significant increase. Another is what the heck is happening? One thing that seems certain is that our data is consistent in showing this result that at this point defies common sense.
We then had tech tools again in the afternoon. We talked a bit more about our posters, learned about Jing and Skype.
I’m puzzled about our data.
Networking meeting: Talked about the meeting tomorrow with the rep from NSF and talked some more about what we wanted to do in our presentations at the symposium. We agreed that each team would present a ten-minute intro to their projects and then we’d split up to circulate and look at posters.
Research: Made three sets of 1.0 mg/ml gels with 100-225 microM FITC. Tested them at 130 and 195 minutes and will continue to test them tomorrow. Unfortunately, the second reading DECREASED in all of the samples, which we thought we’d seen last week, but eventually attributed to the fact that all the samples were still so “noisy” (the peak at 492 nm was nonexistant to minimal, and the background noise was a substantial part of each reading) that they were all effectively zero. However, I was puzzled then by the fact that, in spite of the noise, there were some small peaks that were clearly peaks, and that these data did appear to drop over time. So today’s data shows that same trend (although I know that only two data points is not a complete set of data), so I’m still puzzled, and anxious to see what the data shows tomorrow.
We also made a set of gels at the very end of the day today that we can use all day tomorrow, which will be very helpful–we’ll put it into buffer first thing in the morning, and then can start collecting data on it immediately. Hopefully we’ll get the question of whether the numbers go up (as they should) or down (as they seem to) resolved tomorrow.
Well, at the risk of boring those who don’t like to read about feelings, at the halfway point I am delighted by how much I am LOVING this experience. I LOVE working on something that doesn’t already have an answer. I LOVE the precision with which we need to create, record and discuss our data. I am excited by the thought that next week we might be able to collect real data and really make a lot of progress on our original research goals (answering the “big picture” questions).
As the experience progresses, I find myself thinking of how I can use the need for precision in my classroom. So many of my students are still sloppy in their thinking and in their work. Many of them even think that it doesn’t matter whether they “get the answer” or not, as long as they “know what to do.” In my experience here, precision is essential to knowing whether you have data or junk.
I also want to figure out how to excite them about the fact that, really, people who aren’t in school work solely on issues that don’t already have answers. As students, they are mostly working on tasks and problems for which an answer is known and they just have to get to it. Their focus is on finding the answer and on “getting it right.” Well, when they’re finished with school, there won’t already be known answers in whatever they go into, and they can have the experience to finding the answers to issues or problems that no one knows.
