My project is going smoothly so far, and I’ve acquired a few new friends. There are three Russian students earning their PhDs in my lab. There is also another summer intern working on another project in my lab. All of the Russian students are my mentors and have helped me get through some rough training stages since I’m still pretty green at research having started late May. Perhaps the most important lesson I learned so far is experiments don’t work 90% of the time, and I’ve learned to accept that.
In the last couple of weeks, many PCR’s were done to obtain a good amount of the ERCC3 gene. Specific primers were designed to achieve this. The primers contained attb sites for Gateway cloning. The Gateway cloning system is relatively new and is a much faster and more reliable way to produce clones. The reaction also takes less time than traditional cloning.
After five gels I finally obtained the DNA band that I wanted. There are approximately 2400kbp in the ERCC3 gene. This gene contains a mutation in the base pairs coding for lysine in the majority of pancreatic cancer patients. The goal of my project overall is to get protein crystals of this gene after inducing the mutation by primer design and have E. coli overexpress this gene.
DNA bands of ERCC3 were cut out and then purified using a Qiagen kit, and the concentration of DNA was measured using nanodrop, which is sort of like a spectrophotometer, but instead of inserting a cuvet into the machine, a drop of liquid to be analyzed is put on a little stage the size of a tip of a ballpoint pen.
The concentration was about 26ng/uL, which is enough to proceed with the BP clonase reaction.
The BP clonase reaction involves taking the PCR product with attb sites and inserting these gene segments into another vector called pDNR. This vector was transformed by E. coli, and then the plasmid was purified.
Overall, everything is working so far but on the downside, one of my cell lines perished. Fortunately, all of the cell lines were lysed for DNA and lysates. They were all frozen for backup as well.