Evening Everyone!
Didn’t come into the lab till the afternoon today. Once we got in we removed the buffer from the plugs, and incubated them further in 1X wash buffer for 30 mins, at room temperature. While this was incubating myself and Jo made up 3L of 0.5X TAE buffer, melted agarose and then poured a new gel. The buffer was then removed and replaced with some of the 0.5X TAE buffer, that will be used to run the gel, to allow equilibration. We then cut and loaded 3 samples of fully treated Propionibacterium acnes DNA, 3 samples of Propionibacterium acnes DNA not treated with the restriction enzyme (SpeI) and 1 sample of lambda Hind III as a reference ladder.
Finally we sealed the plugs in with agarose and set the PFGE to run for: 12.7 hours at 6.0 V/cm with a ramping of 1-8 seconds, then 7 hours at 6.0 V/cm with a ramping of 0.1-2 seconds. We haven’t tried ramping before, but we decided to try this set up, as it is the method used in one of the journals we have been working from. Ramping is where one switch interval is used at the beginning of a time run, and another is used at the end, therefore over the time period the ramping slowly changes.
Tomorrow we will be filming our next videoblog, removing, staining and viewing our gel. As mike won’t be in, we will be discussing modifications and a plan for next week on Monday.
See you all tomorrow! 🙂