Evening All! 🙂
Today myself and Jo came into the lab at 9am, and Mike gave us some new stuff to play with! – the newly arrived BioRad plug making kit and restriction enzyme digestion 🙂
We worked through the kit and information provided, made some notes and researched anything we didn’t understand online and through a discussion with Mike. We then produced a new COSHH assessment for our new procedure of making plugs and researched any modifications that journals mentioned when using Propionibacterium acnes specically.
We then made up a stock solution of Chloramphenicol (90 mg/ml), that we will need during plug production. It is a antibiotic, that is bacterial static and stops replication. We will also be using lysozyme, to break down cell membranes, Proteinase K, to break down protein and restriction enzymes, to cut up DNA specifically. Each also has a specific buffer, this is due to enzymes specific nature (pH, Cofactors). We also discussed why it was important to remove remaining EDTA, as it removes cofactors, which are very important to enzyme activity! We also discussed how obtaining the proper cell concentration within the plugs is most likely to be the most difficult part of production. In order to get the most accurate concentration we will be using an OD and CFU/ml against time to produce two sigmoid curves in order to work concentration out.
Excited to start practising these new techniques, so bring on tomorrow morning! 🙂