Week 6: Finalizing Digests, Gel Clean-Up, and Ligation in Preparation for My cGAS Project
Daniel W -
This week, I tackled more research on the cGAS promoter, attended a fascinating seminar on neurodegenerative diseases, and performed crucial lab techniques to move our project forward. Each day presented new challenges, but also exciting progress as we near the final stages of plasmid construction! These experiences will directly prepare me for ligating my cGAS promoter into the plasmid using the same procedures in the coming weeks.
I started the week by finishing my senior blog post and working on the precipitation cleanup we had started last Friday to extract our digested pGL3-basic plasmids. We centrifuged the precipitation solution to complete our extraction, and depending on our yield efficiency (the number of plasmids we successfully extracted from the total), we can proceed with a PCI extraction for the Nrf2 promoter fragments.
The next day, Dr. Carver and I ran a gel electrophoresis test to check the length of our Nrf2 promoter fragments from last week’s PCR and digestion. We got a bright band slightly lower than expected, which suggests the fragment might be incomplete—but we won’t know for sure until we sequence the final plasmid.
Afterward, I calibrated some pipettes from another lab (P20, P100, and P1000) to ensure accurate volume measurements. This process involved weighing water samples, though the smallest pipette (P20) proved tricky as the weight measurement constancy changed due to evaporation. Being precise and swift is key in molecular biology, where even minor pipetting mistakes could affect our results.
Since Dr. Carver was busy with another project, I spent the rest of the day reading, analyzing, and asking questions about my classmates’ senior projects. Later, I participated in our group meeting with Dr. Zhu, where we discussed improving Western blot consistency and undergoing proper training to use a confocal microscope for better imaging analysis. Lei’s project on G3BP1 and STING levels was tied directly to my project on cGAS activation and ferroptosis. I’ll be considering parts of his research as I refine my presentation.
While Dr. Carver was busy, I continued analyzing scientific literature to pinpoint a consensus on the cGAS promoter loci. While I’ve been doing this for two weeks, defining the exact promoter region has been challenging, and I have to double-check each paper as they cite slightly different sequences. This has pushed me to be more critical of research methods and data presentation, ensuring I rely on well-supported findings.
On Friday, I attended a seminar by Dr. Marie Elena Cicardi from the University of Milan, who studies the genetic causes of ALS (amyotrophic lateral sclerosis) and FTD (frontotemporal dementia). These diseases are associated with mutations in the C9orf72 gene, which produces toxic proteins. One of these proteins, poly(GR), accumulates in cells and disrupts normal cellular functions by interfering with RNA-binding proteins that help regulate gene expression. Dr. Cicardi discussed how understanding these toxic interactions could contribute to developing targeted therapies for ALS and FTD. This seminar helped me understand the importance of clear data visualization, structured presentation, and drawing connections between molecular mechanisms and future treatments. I hope I can apply these insights to my research and final presentation.
Back in the lab, we analyzed Nanodrop results from our plasmid and Nrf2 promoter fragment earlier in the week. A Nanodrop spectrophotometer measures DNA concentration and purity by analyzing absorbance at different wavelengths. Ideally, the absorbance ratio (we use A260/A280) should be around 1.8 for pure DNA, but our results were closer to 1.45, which means our solution might have proteins and other contaminants. While this may not impact our final plasmid, it’s something to watch if issues arise later.
To combine our plasmid template with our fragments, we have to perform a procedure known as ligation. Ligation involves joining DNA fragments together using an enzyme called ligase. With the Nanodrop results, we calculated the volume of DNA we needed from the plasmid and fragment solution and the correct DNA fragment-to-plasmid ratio of 3:1. Under Dr. Carver’s supervision, I prepared two solutions: one with both the plasmid and promoter fragment and another acting as a control that didn’t have the fragment. If all goes well, we’ll transform our final plasmid into E. coli on Monday and send it for sequencing by the end of next week.
This week involved a mix of literature analysis, lab work, and engaging discussions on neurodegenerative disease research. Despite small setbacks—like the unexpected Nanodrop results—I’m excited to see how our ligation and transformation turn out. With sequencing coming up soon, we’ll finally determine if the plasmid was correctly assembled. Fingers crossed for successful results next week!
– Daniel
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