Hey Everyone,
Today was a loooong day! Came in at 9am, and as the plugs were still fairly cloudy, Mike suggested we leave them abit longer to allow further lysis, and therefore make the plugs clearer. We made up a fresh solution of lysozyme, proteinase K and mutanolysin, and left the plugs to incubate further until 12pm.
I then began washing the plugs in 1x wash buffer, 1hr each four times. Once finished i washed the plugs in 0.1x wash buffer for an hour twice. I then removed the wash buffer and added speI buffer to allow the plugs to incubate for an hour. After this i added fresh buffer, SpeI enzyme and BSA. The plugs will incubate overnight at 37C.
I made appropriate modifications in amount of lysozyme, mutanolysin, SpeI buffer and enzyme etc. In order to compensate for an increase in DNA conc.
Tomorrow i will be filming my new videoblog, then incubating the plugs in 1x wash buffer, and in 0.5x TAE buffer, before setting up and running a new gel with our new modifications, that looked like they worked very well on weds.