Practicing Protocols

            This week was spent practicing the protocols I learned last week. Christine and all her mentis (now a grand total of four with the addition of the new high school intern) worked on cell culture and harvest with the ultimate goal of extracting protein (MnxDG and EF). This is a long process that involves culturing cells expressing the entire MnxDEFG complex (MnxDG) and those expressing just Mnx E and F. The cells are harvested, treated and pelleted once protein expression is at its peak (when the cultures reach exponential growth), from which the protein of interest is purified. I will be responsible for carrying out the whole protein purification process in three weeks, so it was good to get a feel for what the procedure is like.

We also preformed several PCR reactions to amplify MnxDEF from a P. putida colony, as well as MnxG that contains a His tag sequence and a gene for MnxDEFG. The PCR product was confirmed via electrophoresis. Successful reactions were used for TOPO cloning which were confirmed by a blue/white screen. Ten white colonies from each reaction were verified via PCR/ electrophoresis and patch plated to preserve for future use. I also ran a FailSafe PCR reaction to amplify MnxG with a His tag (Histag_G) from a freezer template and normal MnxG from a colony. I also made buffer stock and extracted plasmids (pTrc with a His tag and pASK) that will be kept as freezer stock for future use, as well as preformed a plasmid mini prep to prepare several freezer TOPO samples for sequencing. I am feeling much more comfortable in the lab and have much more independence while carrying out protocols. I am eager to start my own project but am enjoying the opportunity to gain experience with this diverse array of procedures.