Gregory Nugent's blog

I am finally generating my final data! Every day I am running LacZ assays & reactions in an effort to gather the data from four independent strains for each of my 16 mutants by the end of next week (aka 64 runs). Thus, I am running eight at a time for the next two weeks. This is extremely time consuming since the assays take around 8 hours (sometimes longer) to run and the reaction takes about two hours (which ideally can be ran concurrently with the assay in the same day). Thus, this week has found me working in the lab from around 8am to 7pm (which when my two ho

I have almost generated all of the cell strains with which I will be conducting the LacZ assays on for the next few weeks. It feels great to finally begin to see the fruits of my labor. The only cell strain which isn’t done and frozen is one that was transformed from the troublesome pFS1 plasmid. More specifically it was the strain which was transformed into the ResD mutant, and had difficulty growing on the DSM media – taking an extra day to fully mature. Thus I was only able to get it to the single clone phase before the weekend. I should get

This week definitely had its ups and downs, but I am going to focus on the positives J. I finally have had all four of the plasmids I’ve been constructing come back clean from sequencing! On Monday I had received the news that once again the plasmid pFS1, which was prepared by the highschool intern Felicia, had yet again failed to come back clean from sequencing. Dejected, since I really was hoping I would be successful this time and begin the transformation into Bacillus subtilis with all of my plasmids.   So, we decided to reculture and

This week has been a mixed bag.  It started out well.  I got to begin by learning how to transform the Bacillus subtilis and by inserting my whole promoter sequence (which came back successfully two weeks ago from sequencing) into the wild type strain of competent cells I generated last week.  This involved making DSM agar with Erythromycin as the antibiotic resistance selection agent (since the plasmid will give this resistance to the

I had no mistakes which needed redoing this week! That was a super plus. Also, I amazingly got back my sequence by Monday morning (we didn’t think we would be getting it till like Thursday), and it showed that I had a perfect plasmid (not the case with Keifer – the high school teacher). So for me, that meant I am ready to transform this plasmid into Bacillus subtilus next week.  Before I can do that, however, I needed to make my B.

Yay!  This week went fairly smoothly and without any major surprises (well, for the most part...).  I spent Monday redoing about three days worth of work from week one, all in one day.  Very frustrating that I had to do it, but great practice and I thankfully was successful this time at creating the expression plasmid.  The rest of the week in the lab centered around creating a competent E.