More troubleshooting!!! More life!!!
Back from Winter break, it has been snowing a lot at Bates (not the mention the winter storm Juno) and it has never been this cold in my four years in Maine.
My winter semester has been busy but all the classes I am taking including thesis have been great so I am enjoying it so far. This is my last semester at Bates, so I decided to give it my best shot. This is my last chance to be a college student, to explore, to learn, and to fail in such an encouraging and nurturing environment that I might never have a chance to experience again.
Back to lab work, the fish have been breeding very well. The breeding rate is almost 60-70% of all the tanks we set up. Us thesis students did not have any problem collecting the amount of embryos we need in a few try. At the end of last semester we changed three things in our breeding protocol, including adjusting the light timer in the breeding incubator, and discontinuing using chloro-max and the peroxide disinfectant. These three factors did indeed improve our breeding efficiency.
As for my thesis project, I was faced with a new challenge called primer dimer. All of my PCR reactions show bands around the 80 base pair position, even when in reactions without any DNA template. At first, I thought my reagents were contaminated. To figure out which component of the PCR reagents that were contaminated, I carried out a series of PCR reactions each without one of the components. The results showed that the contaminated primer must have been the cause of the bands, but replacing it with a freshly prepared primer dilution did not solve the problem. Finally, Larissa suggested that I should test for primer dimer by reducing the concentration of primers. It turned out that primer dimer was indeed the problem. I proceeded to adjusting the PCR condition to mitigate primer dimer and optimize amplification of the desired regulatory region of Gapdh.
After optimizing the PCR condition as well as changing the Gel electrophoresis condition, I was able to distinguish the primer dimmer band and the Gapdh band. However, since primer dimmer still occurs, the Gapdh band is very faint, which is not supposed to happen. We decided that it would be best if we design a new pair of primers. So primer design will be my assignment for this weekend. More troubleshooting for me!!!
Bye for now. I will be back in 3 weeks!!!