Describe a time when you had to troubleshoot an issue with a molecular biology technique. How did you resolve it?

One time, while conducting a PCR experiment in the lab, I encountered an issue with non-specific amplification. This was a critical step in my research, as it affected the accuracy of my results. To troubleshoot the issue, I reviewed my experimental protocol and ensured that I was following the correct temperature and cycling parameters. I also optimized my primer concentrations and annealing temperature. Additionally, I performed a gradient PCR to identify the optimal temperature for specific amplification. By implementing these strategies, I was able to resolve the issue and obtain accurate results.

In my previous role as a molecular biologist, I encountered a troubleshooting situation while performing qPCR experiments to validate gene expression levels. During the analysis, I noticed inconsistent amplification patterns across replicates and primer-dimers, indicating potential contamination or suboptimal workup. To pinpoint the issue, I meticulously reviewed every step of the experimental procedure, including RNA isolation, reverse transcription, and qPCR setup. I realized that the problem stemmed from contamination during RNA extraction due to mishandling of equipment. To overcome this, I implemented stricter sterilization protocols and improved my pipetting techniques. Additionally, I optimized the qPCR conditions, such as adjusting primer concentrations and annealing temperatures, to improve the specificity of the amplifications. As a result, I successfully resolved the issue, generated reliable gene expression data, and contributed to the publication of a peer-reviewed article.

During my time as a gene therapy researcher, I encountered a complex troubleshooting scenario with a molecular biology technique called CRISPR/Cas9 gene editing. I was attempting to introduce specific genetic modifications in target cells, but faced challenges with low transfection efficiency and off-target effects. To address this, I performed a comprehensive analysis of the experimental conditions, focusing on optimizing transfection protocols and assessing the impact of varying cell type and culture conditions. I utilized statistical data analysis tools to analyze the transfection efficiency and performed whole-genome sequencing to identify any off-target effects. This analysis revealed that the low transfection efficiency was due to the choice of transfection reagent and the concentration of the CRISPR/Cas9 components. By testing different reagents and optimizing the concentrations, I significantly improved the transfection efficiency. Additionally, I employed bioinformatics tools to analyze the whole-genome sequencing data and identify potential off-target effects. Through these efforts, I successfully resolved the troubleshooting issue, achieved the desired genetic modifications, and contributed to the development of a novel gene therapy approach.