Busy Week…with Much Troubleshooting
The last couple of days have been the busiest by far with days lasting from 9am to 6:30pm and coming in on the weekend Well that’s research for you! Though I can’t complain since most of the time is down-time when I’m waiting for proteins to bind, DNA to replicate, etc. Jack and I have been trying to construct and clone truncation mutants of the regulatory subunit of “protein X” which we believe interacts with our protein of interest, ALK-1. I’ll talk more about the details in my next post). Using a computer simulation program, PDraw, we planned out primers with specific restriction sites that we could use for PCR. While we waited for our primers to arrive, we went ahead and started a restriction digest with a previous aliquot of “protein X”, running a ligation over night, and transforming DH5-alpha bacteria with the “protein X” and vector constructs the next day. Additionally to show me how he had previously confirmed the existence of an interaction between ”protein X” and ALK-1, we started a Western Blot that confirmed the protein-protein interaction between the two molecules through a pull-down assay. As soon as the primers arrived two days later, we dived into conducting some PCR, restriction digests, gel purification, ligation, transformation…But wait we still had to purify the DNA from the DH5-alpha bacteria through a miniprep and run it on a gel to check if it worked alright…Oh and how could I forget, we needed to add the antibodies to our Western blot…essentially by Thursday, we were doing three experiments at once! While it was confusing switching between experiments (I have twelve pages of notes for Thursday and Friday alone!) it saved us lots of time–by the time we finished the first experiment and found it yield unsuccessful results, we could look on the bright side that we hadn’t wasted 4 days just doing that one experiment but still had two others in progress. Unfortunately out of the three experiments only one worked and so this week we are repeating our cloning but slightly tweaking our strategies to troubleshoot. And if we’re lucky, Jack says we might get to start a site-directed mutagenesis where we will get to make mutated constructs of our molecules of interest. I’m going to write a proposal of my research project and post it soon so stay tuned.