S-STEM Scholar Alex Hugenberg-Cox Blog

      At the end of last week, I had tried (and failed) to get a plasmid from Aquaticus. In addition, while going over the lysing procedures over the week I made some adjustments and vastly increased the genomic DNA yield.      Starting on Monday, I want through the lysing procedures. To be honest, not even thinking about it I vortexed the pellet in the special multiwash before letting it sit for five minutes, and vortexed it again before putting it into the lyse. While it wasn't in the protocols I knew it would help break up the pellet and let the multiwash/lyse reach more cells than the pellet. When I realized what I had done, I was worried I had messed it up. Instead we saw the highest DNA yield yet. We  even re-nanodropped it, and checked the nanodrop against other samples to make sure it wasn't broken.          After nanodropping, I did the zippy plasmid extraction kit. However, after running the gel we saw nothing but the ladder, not even a smear or something stuck in the

      In the last blog I mentioned moving onto another project with Kieran, trying to replicate a D.rad lyse onto aquaticus. Before I could start with the procedures I had to make the solutions and create the protocols. It was a lot of dilutions and changing the pH of things, but I also had to make an EDTA solution.       To make the multibuffer I would need, I started by making 100 ml of 50 mM sucrose, and 100 ml of 10 mM tris-HCL. After making those two solutions, I made 100 ml of .5 M, pH 8.0 EDTA. While it was simple enough to combine the ingredients (18.61 g disodium EDTA- 2 H2O and 80 ml water), I then had to change the pH. The original pH was around 3.4, so it took a while, dosing it with HCl to raise the pH and cause the solids to dissolv e. After the EDTA was diluting the 10% Triton x-100 to .1%, which was done when creating the multibuffer. I ended up making 100 ml of multibuffer, with 8 ml of the EDTA solutiom, 5 ml of the sucrose, 1 ml of the 10x Triton x-100, 1 ml of the