qPCR Results

I got the same results as I did before when I repeated the qPCR, which is good in a way because I know that I’m doing the qPCR part right. I got lower standard deviation than I did before but the % input was still about 0.1%. This means that there’s a problem with my ChAP procedure, which is bad because it’s a long procedure and there’s a lot of things that could have gone wrong. Our best guess right now is that there is an issue with the sonication step, specifically that I’m not sonicating enough. I’m supposed to check my sonication but I had to check it on a Saturday and I didn’t know how to do the check. Since no one was in lab that Saturday, I skipped thinking it wouldn’t be that important. I probably should have been more thorough in retrospect. If I don’t sonicate enough, the DNA pieces aren’t as small as we would like them to be. I’m not exactly sure why this would cause my % input to be so low but I have a couple theories. First, right before we do the qPCR, we have to do a DNA purification step to filter out any proteins or cell debris. This step filters out large pieces so if my DNA is too large in size, it could get filtered out which would mean that I wouldn’t get a lot of DNA in the sample that I run for qPCR. The other possibility is that DNA pieces of larger size might not bind to the beads as well as smaller pieces do. This would again result in less DNA being purified and cause my % input to be too low. 

To solve this problem and make sure that sonication isn’t an issue, I’m going to perform a mini experiment where I check how much sonication is required. I’m going to do everything like I normally do in my ChAP procedure up to the sonication point. At this step, I’m going to sonicate for a certain number of cycles to determine how many cycles are necessary to get small enough DNA. This should allow me to see if sonication was an issue and how much I should sonicate in the future.

ChAP Results

So my ChAP experiment didn’t work very well. I didn’t see the result I expected and I didn’t get a very high percent of purification. Previous ChAP experiments have gotten around 0.7-1.0 % of an input sample whereas I got around 0.1%. My results contained a high degree of standard deviation as well which shouldn’t be the case with qPCR. Normally, qPCR produces highly accurate and highly precise results. Since my results weren’t very precise, I’m hoping that I just messed up the qPCR part and not the entire experiment. To check this, I’m going to repeat the qPCR part since I still have left over sample. This should only take me a day or two so it’s not as time extensive as repeating the whole experiment again.

ChAP Start

I started the ChAP work today so I should have results in the next ten days or so.

ChAP Procedure

I’m going to try to explain how the chromatin affinity purification (ChAP) procedure works in as simple, nonscientific language as I can. Before my time in lab, someone modified an existing cell line called HeLa cells so that those cells express a modified form of the protein ubiquitin. Ubiquitin is a small common protein that is used in a wide variety of cellular processes. The protein is modified so that it contains a “tag” of five units of the amino acid histidine. The tag allows us to purify the tagged ubiquitin and separate it from us things in the cell. I’ll get into this more in a second but first the cells have to be prepared for purification. 

The first thing I do in this experiment is to do a gene depletion. The gene we are interested in depleting encodes a protein that we think is crucial in mitotic bookmarking. Without it, genes aren’t bookmarked as effectively in mitosis as they are when the protein is expressed. I do this like I mentioned before using the special HeLa cells that someone in our lab previously made. After we do the gene depletion, we block the cells in mitosis. This is critical because we want to study the effects of cells in mitosis so we need to collect only cells that are in mitosis. To do this, we use a chemical called thymine which stops the cells from replicating their DNA. Then we remove the thymidine and add a chemical called nocadazole which allows the process of mitosis to start but not finish. In this way, we get only cells that are in mitosis. 

The next step is to collect the cells and crosslink them. Crosslinking is a technique that can permanently attach a protein that is bound to DNA. In my case, we have some protein that is bound to DNA and we think that this protein is attached to ubiquitin as well. If we cross link the cells, we can attach the protein, DNA, and ubiquitin complex together much longer than it would be inside a noncrosslinked cell. This is important because it allows us to purify the DNA, protein, and ubiquitin complex via the ubiquitin tag. In future steps, we can reverse the crosslinks and analyze what DNA pieces were attached to protein that was ubiquitinated. 

After I crosslink the cells, I have to lyze them in order to release the DNA which is stored in the nucleus of the cell. We use a couple different chemicals that poke holes in the cell membranes which cause the cells to rupture and release everything that was inside of the cell. The DNA can then be collected by spinning down the sample until a DNA pellet forms at the bottom of the tube. Next comes the annoying part. Each sample has to be sonicated using a probe sonicator that we store in a cold room that’s probably around 35-40 degrees Fahrenheit. I spend close to 30 minutes in the cold room for each sample and I think I’ll have to do four samples so I’m going to get really cold. Anyways, what sonicating does is a breaks up the DNA into much smaller pieces that are easier to work with. 

After sonicating, I add special beads called avidin beads which only bind to the tagged ubiquitin, protein, DNA complex. After leaving this overnight, I can throw away whatever didn’t bind to the beads. Next, I reverse the crosslinks which leaves me with two things: one being the beads which are still attached to the tagged ubiquitin and second, the pieces of DNA that were once part of the complex. I can then collect the DNA and analyze them via a technique called quantitative polymerase chain reaction (qPCR). This process allows people to determine exactly how much DNA is in a sample. In my case, I hope to see a significant difference in the amount of DNA purified between my control sample and my gene depleted sample. 

This whole process takes roughly ten days so it takes a long time to get only a few data points. It’s long work but we should be able to get good results from the experiment and hopefully publish a paper containing the results sometime soon.

ChAP beginnings

I’m beginning to put together a protocol for the ChAP I have to do like I’ve talked about before. We have an old lab protocol for it but it contains a lot of things that I don’t need to do and some things that I need to do aren’t on there so I have to figure out what I need to do. Once I put together a protocol, I’ll send it to the grad student that just left and has done a lot of ChAPs before to make sure that I have everything right. 

Writing this protocol is a unique experience for me and it’s kind of cool. Every other lab experience I’ve ever had, whether it be in general chemistry lab in high school or now in an actual research lab, I’ve had a procedure handed to me and all I’ve ever had to do is follow it and do exactly what it says. This is a bit different since I need to figure out what I have to do and make sure that my procedure actually completes the task. It’s a little bit intimidating to do this for the first time but this is what professional researchers do. It’ll be a big learning experience both in that I will learn the science behind what I’m doing and that I’ll learn a lot about the scientific process. I think this will also make me feel like I’m developing more as a scientist. Before, I’ve been doing pretty basic stuff but writing and performing this ChAP experiment is more of a challenge. I’m looking forward to it.