Determining the Dependence of Nicotinic Acetylcholine Receptor Expression on Opioid Receptors

Welcome to the (probably) penultimate week of my project! This one was tough, as I barely made any progress and my mentor was out of town. On Monday, I completed the last steps of the RNA purification process for the four samples I was trying to obtain. This was, however, my first time performing the entire procedure from start to finish. That might explain why, on Tuesday, the gel results were disappointing, to say the least. Only one out of the four RNA sequences was verified, so I had to redo the procedure for the other three. That day, I attended a conference about using a noninvasive electrical headband for performance enhancement (sounds like sci-fi, doesn't it?). On Thursday, I started to do exactly that-- but I hit a snag. The gel after the very first PCR (performed just to "check" that the process is on track) failed. This was extremely strange, as I had already done this step successfully in the past. I did it again-- still nothing. I hadn't (cons

Hey, everyone! This week, I got to utilize some interesting software in addition to the biotechnology in the lab. I spent Monday finishing up the FastCloning protocol for two final DNA sequences and sending them to the sequencing lab. In addition, I re-measured the concentrations of the DNA I had been trying to convert to RNA (using a spectrophotometer)-- it turns out my initial measurements had been way off, which might have been causing the issue. I also learned how to use a DNA chromatograph viewer called Chromas, which is how we manually "fill in" any bases that the sequencing machine fails to recognize. The various peaks indicate different bases, or "letters" of DNA On Tuesday, I reran the now accurately measured DNA samples through the PCR machine and found success when I ran a gel afterwards. Unfortunately, as luck would have it, just one of the samples failed-- the second one from the top, which has two distinct bands instead of one.  Fi

I got right back into the swing of things this week, and learned how to perform even more procedures as my project progresses. On Monday, my mentor told me that he had already gone ahead and sent in the previous week's samples for sequencing (the final step of the cloning process) during my hiatus. The results hadn't arrived yet, though, so I instead practiced the next step: in vitro  transcription. Although DNA is the ultimate blueprint for making proteins and therefore organisms, it is not actually directly translated into them. That role belongs to an intermediate molecule known as RNA, which is produced in the cell's nucleus using DNA as a template. After RNA is translated into proteins, it breaks down, halting the production of proteins but leaving the original DNA unaffected. What we had at that point was DNA; injecting it into the egg cells would do nothing, as the cell would continue to only express the DNA in its original genome. However, RNA  would be i

Hey, everyone! Short post this week, as I only went in on Monday and took the rest of the week off for spring break. I retrieved the plate that I had inoculated on Friday, which now had several bacterial colonies visibly growing. If you remember, these bacteria contained the one type of DNA which hadn't worked last week. I amplified that DNA using PCR, which took up most of the day. I then ran what is hopefully the last-ever gel on the products and, to my relief, the results were successful! Now that we finally had all the DNA we needed, I chose two colonies from each plate (based on previous gels) and cultured them. The last step will be to isolate the DNA and send it to another lab for sequencing, which will serve as the final confirmation. See you next week!

I nearly finished the cloning process this week but ran into my first bout of trouble. No project would be fun without a challenge, though, which is exactly what I encountered. Most of the week was spent diagnosing then attempting a solution-- whether it works remains to be seen when I return! -------------------------------------------------------------------------------------------------------------------------- On Monday, I ran a gel on the DNA that was sitting in the fridge over the weekend (if you haven't noticed, I do a lot  of gel electrophoresis, as the DNA has to be verified as correct after every step of the FastCloning process). Oddly, nothing showed up in the gel. I checked everything about the gel setup itself (including the agarose solution, this time), but nothing was wrong, meaning the DNA was the culprit. Dr. Chang suggested that I amplify the DNA-- maybe the issue was that there simply wasn't enough of it. I spent the rest of the afternoon preparing t

I got right back to work this week after my mentor arrived from Minneapolis on Monday. The final DNA sequences had finally arrived while we were out, so we began the process of molecular cloning: inserting the sequences into plasmids (larger, circular DNA fragments that carry the desired sequences), coaxing some bacteria into taking in and naturally reproducing the plasmids, and finally purifying the "cloned" DNA product. This can be done in a variety of ways, but many traditional methods involve expensive kits or have an unacceptable risk of mutation. So we used FastCloning, an inexpensive and effective method that my mentor developed and published in 2011. It carries that name because it is faster than the aforementioned methods; however, as I was about to discover, it is still very time-consuming (at least with my lack of experience). This one's a bit of a doozy; consider it as a supplement to last week's incredibly short one. ---------------------------------