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.