Evening all!
Myself and Jo spent this morning researching the next stages of our project, then we came into the lab for 3pm, in order to take our gel out of the PFGE. We then stained it up and viewed it with Mike. We saw much better separation than before, with the bands reaching all the way to the bottom of the gel, and running off. Both yeast samples, BioRad and NEB, also appeared consistent with each other. The 1Kb ladder was not visible, which was expected. However, the separations slightly shifted to the right, producing wonky bands, that unfortunately weren’t very defined. Mike said this was most likely due to imperfections in the agarose, and we could rectify this by producing 150ml of agarose in order to reduce error. In order to try and stop the yeast sample running off the gel we decided to change our switch interval as well. We also decided that as the NEB yeast has been the most consistent and successful sample, we would stop running the BioRad yeast and 1Kb ladder. We will keep the size of the plugs the same, and well as sealing them with agarose.
Reviewing the NEB yeast information we decided to try running our next gel following the guidelines provided, 1% agarose, 15 hours at 6 V/cm with a 70 second switch interval and then 11 hours at 6 V/cm with a 120 second switch interval. Using 0.5X TAE buffer, 2 samples of NEB yeast and at 6°C as before.
See you all tomorrow morning as we set up our next, and possible last gel before starting on our next stage in the project! 🙂