Third Time Isn’t Always the Charm

Why is my lysate so much more yellow than everyone else’s?

Pretending to not panic, I casually checked out all of the other lysates in the rack. They were mostly clear, some with a faint yellow. Yet, there mine was, a distinctively darker yellow.

As I retrieved my lysate and went back to my bench, I noticed that I did not have any leftover phage buffer from last class. In order to collect the lysate, I flooded my webbed plate with phage buffer, allowed it to sit, and then collected it into a separate conical tube. At least, that was what I was supposed to have done. In place of where I thought I would have my leftover phage buffer was a tube of yellow enrichment broth, and my heart sank.

I had flooded my webbed plate with enrichment broth rather than phage buffer.

Since I already threw away my round of plates that were from serial dilutions, I had to perform serial dilutions and plaque assay again.

The number of times I have messed up in this lab is more than I would like to admit, and it seems as if I had a talent for making mistakes at every opportunity. However, as the end of November loomed upon me, I realized this was no laughing matter. I was running out of time, and just when I thought I had my lysate and could progress along, I had to mess up.

There were no alternatives – with all my materials gathered once again, I performed serial dilutions and plaque assay, followed by flooding the webbed plate. I ensured to add all the right materials, and finally, I got my lysate. I thought the worst was over, since I only had to obtain my EM images and extract DNA next. I found comfort in the fact that I will only be getting results from here, and ensured to be extra careful in extracting my phage DNA in preparation for gel electrophoresis.

Some time between the first and second round, I handed over some of my lysate to (hopefully) obtain EM images. Four other classmates and I went at the same time, and I was the last to get my image since I spent the first part of class obtaining yet another DNA sample. As each person got their EM images successfully, my apprehension of not getting anything concrete only amplified exponentially. My heart raced as the professor proceeded to examine mine, and the encompassing darkness only contributed to the tension.

“Look, another Myroviridae!”

Words could not explain how relieved I was to know that I did get something. I was, once again, hopeful for the future of my phage, only to be disappointed by my lack of DNA data from 3 rounds of gel electrophoresis.

To briefly summarize my dismay:
– 1st time: no band
– 2nd time: no band
– 3rd time (even after the TA’s tried DNA precipitation over Thanksgiving break): no band

Fortunately, there were many others in the same boat as me, and Dr. Fisher told us that the purpose of getting DNA was really only for whoever will be sequencing his or her phage. While I was slightly disappointed initially, looking at my EM images reminded me of how far I have come in both contributing to this SEA-PHAGES project and in cultivating my scientific self.

em_420-33-1

The EM image of Phomnie (Phomnie is the name of my phage)

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