Hello All,
Unfortunately the re cultured samples hadn’t grown enough on Monday to use, so we left them another day and night until Tuesday. Today, although the growth was minimal we decided to start plug making. We added chloramphenicol to the samples, left them for an hour, and then centrifuged them all down until we had appropriate sized pellets. However, some of the samples did not readily form pellets, and several pellets were much smaller than others or were very dark in colour.
We then combined the pellets with cell suspension buffer, shook them to combine and added agarose. We then pipetted the samples into plug molds and left them to solidify in the fridge. Once solid, we pushed each plug into a separate eppendorf, added appropriate lysozyme, buffer and mutanolysin. However one plug hadn’t soldified enough and was discared. They are now incubating at 37C. At 6, i will remove the lysozyme, buffer and mutanolysin, and add proteinase K and buffer. Incubating them overnight at 50C.
Tomorrow we will be washing the plugs, and incubating them in restriction enzyme, buffer and BSA.