plug molds – SB209

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.

Good Evening Everyone!

Busy day today! Myself, Jo and Mike discussed possible modifications to the plug making method when using Propionibacterium acnes. We decided that we would need to use mutanolysin, which aids in cell lysis, as Propionibacterium acnes can be difficult to break into! We also decided to prolong the incubation time after the addition of mutanolysin and lysozyme from 2 hours to 3 hours at 37°C, in order to give them plenty to time to work on the Propionibacterium acnes.

We then began running through our first attempt at making the plugs! We started by adding chloramphenicol to our Propionibacterium acnes TYGE broth (that Mike inoculated for us). Once that had incubated for an hour we centrifuged it at full speed for 5mins, until we obtained a pellet. We then eluted this pellet in cell suspension buffer. We melted agarose and added these together. We then pipetted this into plug molds, which required a bit of practise! (we made 10 plugs) and left them at 4°C, to solidify. Once set we removed them into a universal tube and added lysozyme, it’s buffer and mutanolysin, it was then incubated at 37°C for 3 hours. We then removed the plugs and rinsed the plugs with pure water. We then added proteinase K and it’s buffer and left it to incubate at 50°C, until tomorrow morning.

We will be washing the plugs A LOT tomorrow! 4 times per plug 1 hour each, with wash buffer! We will then be working through the restriction enzyme digestion of the plugs info, and hopefully get ready to set up our first run with our hand made plugs! 🙂

See you all tomorrow!

Tag: plug molds

Hello All Unfortunately the re cultured samples hadn’t…

Good Evening Everyone Busy day today Myself Jo…