Peddie EXP 2017

Week 4 and 5 were mainly focused on the same thing, transforming bacteria.  Since last week, the Y2F plasmid was not cut properly, yielding many bands of different sizes, rather than a band around 6k and one at 350 base pairs long.  We first double checked the plasmid map to make sure that the enzymes that we used were in fact the correct ones.  They were, so we contacted the company about the improper cuts.  They did their own tests and found that the enzymes were manufactured improperly.  They remade the construct and sent it back.  This time, the proper bands appeared.  We then had to send these bands out for sequencing to make sure that the inserts were in fact the mutated form of the Y2F insert, since the mutant and wt versions are the same length.  The image below shows how sequencing is done. The sequencing for the CAR19 with a Y2F insert came back with the results that we expected, the bands matched the mutated version exactly. ...

So these past two weeks were extremely busy, and quite eventful as well. Most of the week was spent trying to transform DH5a competent cells with specific Prdm16 plasmid we made using Gibson Assembly. We tried increasing the concentration of plasmid, using new Gibson Assembly master mix, and even tried to run a positive control to make sure everything was working. All of our attempts failed, indicating either the cells or the master mix was the problem. So I transformed pure plasmid into the DH5a cells to see if these would transform, and they did...sort of. There were indeed bacterial colonies, but our transformation efficiency was 100x lower than what it should have been. This obviously was a problem, so we ordered new, ultra-competent bacteria to see if we could at least transform those. And indeed, our Gibson Assembly worked. All total, we spent about one-and-a-half weeks just trying to get the transformations to work. However, that was not the only project we did. In addition to...

For this week I was tasked with decommissioning a past lab so that the Capone group could start to move into the new space. Currently the Capone accommodation is rather small, so the new lab will definitely help with space issues and also help with tons of new equipment that is in this lab.  This is a picture of the new lab. The first step was to contact hazmat and have them clean all of the fridges and freezers that were in use, and to remove all potentially dangerous chemicals. Next it was my job to take a heat gun and start to defrosts ice that has accumulated over 7 years of continuous running.  We are scheduled to almost be done, and just recently hazmat has started to come in again, and begin the task of disposing all mystery chemicals and items that are potentially dangerous to the people who will be working in the lab.  During the time of decommissioning the lab I have also been working on my project of homogenizing the samples th...

As one may infer from the title of this post, things have not gone as planned during the past week. I discovered last Monday morning that majority of my algae cultures were unfortunately contaminated. For half them, or rather all the cultures for one species of algae that I was growing, they were cross-contaminated with algae of a different species, that was being grown in the other three cultures. Of the remaining cultures that were not cross-contaminated, two were instead contaminated with microorganisms that originated from the last culture. As such, I have to restart my experiment. However, instead of using the exact same species of algae, I will be swapping out one of them for another species of algae. Restarting the experiment is going to take a few days though, as I will be reusing the bottles that I had used to cultivate my previous batch of algae. But, I have been given permission by my PI to carry out the experiment as planned at the start: cultivate the algae for som...