Emily Guo, Week 6, Almost the Home Stretch

After dealing with plasmids and cell culture for most of my time here, I was finally able to witness some bisulfite conversion! This process essentially converts unmethylated cytosines to uracil, which, when the DNA is sequenced, allows us to determine methylation pattern. Unfortunately, the first time we ran the gel for the PCR amplification product yielded no bands, and the same was the case for our second gel. Even our positive controls did not show any DNA the second time around. At this point, we had to go back and redo the bisulfite conversion (as we were also out of DNA) and find out what went wrong during the whole process of bisulfite conversion, cleanup, and PCR amplification. We also tried a different bisulfite conversion kit to see if either the QIAGEN (what we initially used) or the ZYMO bisulfite conversion kit worked better. And after we ran the final gel on Friday... we still had no DNA. We will have to figure this out next Monday.

Sad image of a gel with just the 100 bp ladder

This week, we’ve also taken a look at our serial dilutions (from a few weeks back) to see if we had any cell growth in the 96-well plates. Many of the wells didn’t have any cell growth at all (the media was still a reddish-pinkish color); I would say that we had about 12 wells turn yellow per each 96-well plate. This was much less than we had calculated for, and Alyssa said that maybe the cells just ended up clumping together instead of being dispersed evenly across the wells. Another reason could be that the cell lines were just not good at surviving these single-cell dilutions (online, many people had suggested using conditioned media for cell lines, which is essentially filtered “used” media containing all the stuff excreted by the cells). But because it took about a week longer for these cells to grow compared to Arun and Alyssa’s previous dilution, it could be a good sign that we indeed have one cell per well.

Our other well plates, which we mistakenly calculated for 10x the amount of cells, had much more yellow wells, but we’re thinking that these wells all probably contain multiple cells. After we transferred the cells from the 96-well plates to proper wells, Alyssa decided to keep the 96-well plate just in case.

Me transferring the cells from our serial dilutions to a 12-well plate.

As for electroporation to deliver plasmids to our cell lines, I might not be able to see it happen while I am at the lab. If we were to electroporate cells now, there would be a ton of new cells to take care of, especially after serial dilutions. And taking into account the cells that we are currently keeping alive, there would be too many cells for Alyssa to take care of by herself once Anna and I leave the lab. It just wouldn't be the best idea at this time, especially with the move looming over everyone’s head.

Overall, my week comprised of a lot of cell culture, gel making, PCR amplification, and DNA extraction, as well as some bisulfite conversion. All the while, many people have been coming to our lab to discuss the move (how to transport certain substances/samples, the storing of equipment) including the lab safety guy whose safety class I attended back in June! I have to admit, it feels a bit like things are coming full circle. Moving boxes are popping up, and we haven’t seen Dr. Tycko for a few weeks now as he's doing whatever he's doing in Hackensack. I have a feeling that next week will be interesting as moving day gets nearer and nearer.