2 months into the semester
We are 2 months into the semester now and thesis is going fine. There are still lots of troubleshooting to do but so far I am enjoying it.
Since the last blog post, I have been testing the procedure modified by alumnus James Meyo. I ran to more problems than expected. The first problem was that my DNA degraded before it should. When we ran a gel after crosslinking to test the fragment size, we saw lots of smearing and the absence of genomic DNA. The other problem was that I got foams from sonication, which indicated uneven fragmentation.
Last Saturday, it was very nice of Larissa to come in lab and work with me through the procedure to make sure I was doing it correctly. She showed me many tips to enhance embryo extractions, as well as helped me figured out that I used the wrong concentration of Trypsin, which made my DNA degrade before it should. Secondly, as simple as the sonication procedure look, it actually takes lots of skill and practice. I spent the afternoon with practicing that skill under her instructions. Larissa brought her cutie dog Bella and we had fun playing with her. We also enjoyed our chats about dogs, food and living in Maine.
Another theme of this week’s lab is positive control. Not until last year in our molecular biology lab that we formally discussed the role of positive control in scientific studies. Positive controls are treatment replicate in which we use biological components that were found to successfully interact with one another and yielded positive results. Positive control ensures us that our experimental procedure was carried out correctly, and that we can make convincing conclusions regarding our hypothesis regardless of our experimental results. In this case, we must use a proteins that were found to bind and activate transcription of a known gene, and such interaction was successfully tested in previous studies. After some preliminary searches, we chose Histone 3 that are mono methylated at Lysine 4 (H3K4me1) since this genes up regulates the expression of many ubiquitously expressed and transcriptionally active genes. From the paper by Aday et al., 2011 that focuses on the binding profile of H3K4me1, we found gapdh, a gene that is expressed from zygote to adult stage, and was found to be activated by H3K4me1. Such interaction was successfully tested in Aday et al., paper so we decided to use H3K4me1 and gapdh for our positive control.
Next week, I will be working on my sonication skill to ensure chromatins are evenly fragmented to 200-500 fragments. It everything goes well, I will proceed to testing the Immunopreciptation procedure and the positive control. We are hoping to have the positive control procedure finished by the end of this semester.
On that note, happy Halloween!!! Also, happy first day of daylight saving time and happy first snow of the season!!!