Evening all!
Today consisted of A LOT of waiting! We started by washing the plugs in 1x wash buffer 4 times, for an hour each! This is to make sure that all the lysozyme and mutanolysin was removed. The plugs could then be stored in 1x wash buffer at 4°C.
We then put each plug into a separate eppendorf tube and washed once with 0.1x wash buffer for an hour, and then once more to ensure all EDTA had been removed, as it will remove cofactors that are very important to the restriction enzymes we want to use next! We then removed 3 plug eppendorfs, and put them into the fridge, in order to compare samples that were treated with restriction enzymes and those who weren’t. We removed the remaining buffer in the other 7, and added 1x restriction enzyme buffer to each, and left for an hour, at room temp. with gentle agitation. After this, we removed the restriction enzyme buffer, and added fresh restriction enzyme buffer, restriction enzyme (SpeI, which cuts at ACTAGT) and BSA (which acts as a crowder to increase conc. and allow increased collisions). This is to allow the fragmentation of our Propionibacterium acnes DNA, which is on average 100kb size fragments. We then incubated the plugs overnight.
Tomorrow we will be finishing off the plugs, preparing a new gel run and running a new gel with our home made plugs and new modifications.
See you all tomorrow! 🙂