Novelty in the Making
Here we are with only three weeks left (this week included) and I am having some scientific difficulties.
My colleagues and I are all very tired and stressed, but we are pushing through. Ken and Marcus have been diligent in finishing up getting qPCR results for their housekeeping genes. Hadley has been working on getting data for her last couple genes as well. We all have different projects and are by far not experts on each other’s topics, so please go and read their personal blogs too! Anyway, last Friday I had to essentially re-run all of the mini experiments I had done to date. This was due to many failed attempts at ligating my gene (Biliverdin reductase b) with my TRAP construct. Ligation is the process of me trying to reclose my vector after manipulating it to have my gene inside of it along with the already present zebrafish ribosomal protein (rp10) and enhanced green fluorescent protein (eGFP). This is necessary for the next student to be able to microinject the fish with the completed z-TRAP transgenic.
This past weekend I ran gel electrophoresis on the original PCR product (blvrb with GATA 1 binding sites inside the sequence), which still gave me a clean single band at the desired fragment length. I also ran a gel of the PCR product post purification to ensure DNA sample remained after stripping the solution of enzymes, nucleotides, primers and buffer components from the kits I had to use during these processes. Finally, i was able to re-digest my PCR product as well as my TRAP construct.
A digestion in this scenario is not me eating the material and breaking it down (it’d probably kill me), but instead it is the process of adding restriction enzymes to genetic sequence. Since I am cutting open a plasmid and adding things, I need to be sure that the two ends of the circle don’t close back in on themselves before I can get my stuff inside. My restriction enzymes of choice are BamH1 and Nhe1 because these both work well together with 100% efficiency in buffer 2.1. Most importantly though, the stretch of Blvrb that I amplified in PCR does not have smaller chains of bases that are recognizable to either of these enzymes. We wouldn’t want them to just cut the construct in a willy-nilly fashion.
I am about to do another ligation step in hopes that it works this time, but science is tricky. The temperature that the ligation happens in, the amount of digested and purified PCR product, as well as the amount of digested and cleaned TRAP vector for a successful z-TRAP transgenic are all unknown so this phase is trial and error. My advisor has given me a few different sets of conditions that work in standard ligations of plasmids, but this is just a start. I will plate my ligation products on ampicillin plates using heat competent E. coli cells. The bacterial colonies will only grow in the presence a successful ligation. This is due to the plasmid containing specifically an ampicillin antibiotic resistance gene that the bacteria can actively incorporate into their genome, allowing them to survive and grow on the plate with the otherwise deadly antibiotic. I am also preparing to lead my journal club presentation (next Friday), turn in my PowerPoint for my Mount David Summit talk (next Friday), and finish writing my actual thesis write up because we all bind April 5th! Wish me luck guys!