Final Thoughts

My STEP experience in undergraduate research this summer was a great experience for me. It taught me a lot of things not just about lab techniques but about the direction I want to take my life. One of my goals this summer was to find out if I wanted to go to grad school and become a researcher or if I wanted to do something else. I found out that while I like doing research, I don't think I want to do it as a career. I don't like the fact that I don't get a lot of personal interaction with other people outside of my lab. I think I would prefer becoming a doctor or something like that where I have a lot of personal interactions.

As for my research, hopefully our paper will get published sometime soon. In addition, we are going to try to identify the protein that sits on DNA at the promoter region. We're not sure how difficult this is going to be but we're going to give it a shot. If we can do it and characterize the protein, it'll be a major paper that would likely get published in a major journal, which would be a big deal for my professional career, even if I don't go into the research field. But that's a long way down the road from now.

Overall, my experience has been a great one. I would like to thank STEP for giving me the opportunity to work full time this summer in a research lab so that I could learn all of the things that I did. It was a summer where I learned a lot about myself and what I want to do with my life, which is a great thing for a third year undergrad. Hopefully, I'll continue to learn many things in the lab and about myself so that I can accomplish all of my academic and personal goals.

Thanks for reading!

Final Results!

This time I did the two repeats at once. It's more work in the short term but I'll be done faster this way. It's not that much work with the cells but the cell lysing and sonication take a lot longer. I staggered that a couple of days apart so it wasn't that bad.

Both repeats showed the results I'm looking for though. It's not great but I think it should be good enough to go with. Again, the new siRNA didn't show the effect as well as the old one did but I think we proved that the result wasn't just a fluke with the old siRNA, which was my whole purpose.

We're ready to move toward publication now. Most of the paper is already written but we'll have to add a little section about my results and a figure displaying my results. It shouldn't take too long for the paper to be submitted for review. Once it goes out, it'll be reviewed by a few people in the scientific community. They'll make a few revisions and suggest some experiments that we need to do for the paper to be approved and published. Hopefully it won't be too much of a hassle and it'll go to publication quickly. We should get the reviewers comments sometime in December or early January. We hope that it'll get published in the spring some time.

I'll do a summary of my STEP experience in undergraduate research in the next few days. That'll most likely be my last post so it'll be a good one.

Result!

Finally I got something useable. Both siRNAs finally showed the result we were expecting. The new one I am testing didn't work as well as the old one but it doesn't really need to. We just need to show that the new one works okay and that the result we got with the old one wasn't some kind of fluke.

So this is really good news. Hopefully I'm almost finished with this project. I don't like how long it takes me to get one piece of data. But hopefully I only have to do two more repeats and then I'll be done. We have to do repeats to ensure that we get data that is statistically significant. There's a complex statistical test that we use that I won't go into but basically the more repeats we get, the more likely that the results will be significant and accepted by the scientific community.

Frustration

Well, I was expecting this one to work but for some reason it didn't. Again, I don't know why. I used less cells this time which probably wasn't the problem. This time, the problem was that I got contamination again from somewhere. I still have no idea where it's coming from. Nothing should be in my final sample except the DNA I'm interested in. It doesn't make sense. It's very very frustrating.

In the hopes of eliminating any contamination, we're going to add another purification step. This step involves using nickel beads, which are very similar to the avidin beads I've been using. The principle idea is the same. The nickel beads will bind to the tagged ubiquitin and everything else won't. Then after we wash the beads a couple times, we break the bonds between the nickel beads and the tagged ubiquitin and capture whatever has the tagged ubiquitin protein. Then I'll take that sample and apply the avidin beads like I have been doing. We're hoping that adding a second purification step will eliminate any contamination. We'll probably get less yield as a result of doing another purification step but we should have enough DNA to do the qPCR and get decent results.

That's the theory at least. But nothing much else has worked for me recently so I've learned not to get my hopes up.

Finally Got Some Good Results

The double depletions did the trick. I got the results I expected with one of the siRNAs I was using. However, the new one I was using, the important one, didn't show the same results. I think I know why this is and I don't think it's a major problem so the results from this experiment are good news. Basically, my mistake was using too many cells. If I use too many cells, my siRNA will basically get diluted and not work as well as it should if I use less cells. So the only thing I have to do is use less cells next time and I should see the result I'm hoping for. Things are finally going in the right direction.

More ChAP Results

Once again, I didn't get the results I wanted. The ChAP worked again but I did see the effect I should have. I don't really understand why because the ChAP is working fine and I've checked the depletion with a western blot so I'm not sure what's going on. It could be that I'm not getting enough depletion. It's possible that there's still enough protein in the cells in order to accomplish the cell's function normally. This is probably the most likely scenario so I think I'll try to fix that first. I can essentially do two rounds of depletion. It's the same technique and procedure, I'm just doing it twice. So it'll take a couple days longer, which makes a long experiment even longer but that's what I'll have to do. Hopefully this will get rid of almost all of the protein in a cell and I'll see the results I'm supposed to.

ChAP with New Cells

I finally have something that I think works well – my latest ChAP finally works well enough to get data from. I still have a few concerns about it but it’s good enough now that I can move on to doing the actual experiment I was supposed to do, which is a ChAP with gene depletion. I’m hopeful that when I do the gene depletion the ChAP still works. Gene depletion shouldn’t affect the ChAP process but I’m a little worried that my latest results were just a fluke and that I haven’t actually solved any of the problems I’ve been having. I’m not super positive about it because a lot of things have gone wrong this summer with the ChAP experiments. 

It’ll take me a while to get these results since I’ll be going on vacation for a week and half. But hopefully everything will work when I get back.

Tagged Ubiquitin Check

I finally got the results to check for the tagged ubiquitin and, of course, things aren’t that clear. Both my new cells and my old cells showed some tagged ubiquitin with the new cells showing slightly more. This is a slight problem because I was hoping that my old cells would not have shown any tagged ubiquitin, which would have definitely give me the answer as to why my experiment hasn’t been working. The fact that tagged ubiquitin was expressed in the old cells worries me a little because it could mean that there’s still something wrong with my ChAP procedure.

The problem with this check though is that I have no control. The main problem is that I don’t know how much of the tagged ubiquitin I should see. I could be getting a lot of tagged ubiquitin or not enough. The only thing that I know is that it’s there in some amount. It’s frustrating because the tagged ubiquitin is the most crucial part of the experiment but it’s hard to tell if it’s right or not.

My plan for the next week or so is to do another mock ChAP. I’m hoping that the change of cells makes the ChAP work this time. If not, I’m not sure what I’ll do.

2nd ChAP Results

My second real ChAP results were a little bit different but still not correct. I did achieve higher levels of % input, closer to what they should be. The problem is that my negative control, a gene called IL2, was also very high. A negative control is used to show that things you expect not to work don’t actually work. It’s a way to avoid getting false positives. In my case, I likely got a false positive because my IL2 was high. In the type of cells I’m using, IL2 is not expressed so you would expect to see a low level of IL2 bookmarking. Since I observed a higher IL2 amount than I should have, something went wrong with the experiment and my results aren’t reliable. This is a different kind of error than what has happened before. We think that there could be something wrong with the cells since IL2 bookmarking was so high, which shouldn’t be the case. However, my % input was higher than normal, which I’m having a hard time of explaining. 

There are a couple things that could be wrong with the cells. First, the cells could just be old. Although the cells we are using grow forever, we do find that weird things start happening when we grow them for a long time, such as the expression of a gene that is not normally supposed to be expressed. This is a possibility and we don’t have any real way of testing this. To eliminate this possibility I’m thawing new cells that I know come from a stock that work. The other possibility that I can think of is that the ubiquitin tag isn’t being expressed that well. Again, I have no idea why this would be but it would explain why I’m not getting a very high % input. We can test this however. What we can do is run a western blot and use an antibody like molecule that binds to a molecule called avidin which binds to the tagged ubiquitin (the antibody like molecule is actually a protein with horse radish peroxide that binds to avidin). We should see a streak at the top of the gel, indicating the presence of tagged ubiquitin. 

In this case, I’m actually hoping that we find that I’m not using cells with tagged ubiquitin. Although it would mean I had wasted a lot of time with a simple issue, I would finally know what was going wrong with my experiments and maybe finally finish this project.

Mock ChAP

The results of my most recent ChAP attempt (I’m calling it a mock ChAP because it’s just an attempt to see if I can get the experiment to work right) weren’t so great. I got more of the same results. My % input was again too low. Since I checked the sonication and the experiment still didn’t work, the sonication isn’t a problem. I’m not sure what the problem is now. There are a couple things that could be going wrong with a couple of solutions I’m using. I suppose that it’s possible that some of the solutions I’m using are bad so I’m going to remake some of them. Other than that, I’ll have to be extra careful that I’m doing everything right and following the procedure exactly as I’m supposed to. I’m not sure what else could be going wrong at this point.

Sonication Check

Here are the results from my sonication experiment. This picture shown is the result of running the DNA samples on an agrose gel. Agrose is a type of sugar that polymerizes into a gel. In order to run an agrose gel, you have to load a small amount of DNA into the gel and apply a voltage through the gel. DNA is negatively charged so when the current in applied, DNA will move through the gel toward the positive terminal at the other side of the gel. Inside the gel, DNA pieces of different sizes will move at different rates, similar to how proteins move through a gel in western blots. Larger pieces of DNA move slower than smaller pieces of DNA which allows us to determine what size DNA fragments we have in our sample. 

Let me explain the picture a little bit. The far left and right columns are called DNA ladders. They contain DNA pieces of known size and serve as a marker for how far a specific piece of DNA should run. The bands at the bottom are smaller in size than the bands at the top of the gel. I should also mention that the darker the band, the more DNA there is in that particular location. Going from left to right in the other columns are the DNA samples that I took after I sonicated. Again from left to right, the samples are as follows: 3 cycles, 6 cycles, 9 cycles…up to 24 cycles of sonication. (A cycle of sonication is 30 seconds in the sonicator and one minute on ice). As you can see on the gel, in the samples where I sonicated less, the DNA is larger where as in the samples where I sonicated more, the DNA is in smaller pieces.  The results also show that I should be doing about 15 cycles of sonication to achieve small pieces of DNA.

When I did my first ChAP, I only sonicated for three cycles, which probably wasn’t ideal. However, I’m not sure if the DNA is large enough to cause any significant problems so I’m not sure that the sonication is that big of an issue in my ChAP procedure. This is frustrating because I don’t think that I have a good answer to what went wrong. Since I have the DNA already prepared, I think I’m going to continue with the ChAP protocol to see if the sonication helped to achieve a higher purification. Hopefully it works better this time, but if it doesn’t it means that sonication isn’t the problem that’s causing me to get bad results.