Four weeks of progress
Today is the last day of class for this semester. WOW time flies!!!
One week after the last blog post, I figured out how to obtain the desired fragment size from sonication. YAY!!! Once I got a hold of the basic technique, I quickly moved on to working on the positive control. Before all, I have to test the efficiency of the primer pairs obtained from the Aday et al. paper. The result turned out great. I got 100 base pair fragments, which was as predicted using BLAST.
The next step of the procedure is Immunoprecipitation (IP). I am using an IP technology developed in the 1980s called Dynabeads. Dynabeads are magnetic beads that have binding sites for antibodies, which will bind to our antibody of interest (anti-H3K4me1) upon incubation. Addition of antigen (H3K4me1) allows the protein to bind to the antibody, and the Dynabeads-antibody-protein complex is easily separated out of the solution by using magnet. The magnetic separation is gentle, reduces variability and yields more reproducible result.
The DNA bound to H3K4me1 is then reverse crosslinked and PCR with the aforementioned primers to see the present of bands. After the first try, I got quite low DNA yield, but was still able to get a bright band after PCR and gel electrophoresis. I tried the IP procedure again; this time I also included a negative control for which I did not use any antibody. The result showed up great; the negative control showed a very faint band on the gel, which was mostly attributed to background noise, and the antibody treatment showed a nice bright band. For the rest of the semester, I will repeat the IP procedure once more. This time, I will include the two aforementioned treatments, but also add the input (positive) control, and a negative control for which I will use a non-specific antibody.
Beside lab work, I also got the first draft of my introduction done before Thanksgiving break. Larissa kindly provided some feedback on the writing and showed me how to position figures and tables in the paper. Now I am working on the Materials and Methods and Result sections!!!
Next week is finals week. Best of luck to everyone !!!