S-STEM Scholar Carly Ziegenhagen's Blog

Introduction This week, we ran a gel electrophoresis on our amplified DNA samples of D. aquaticus from the PCR reaction ran the prior week. The fragments we used were intended to be 7kb long, so the expected result from the gel electrophoresis was two bands measured at the 7kb mark of the ladder. This result would indicate that we were able to successfully isolate the 7kb plasmid. Results The results from the gel electrophoresis showed areas where the dye was smeared across the gel, and no visible bands could be found at the 7kb mark. Bands were visible at two other spots, much lower than 7kb. Discussion These results indicate that the DNA sample isolated is much shorter in base pairs than we intended. The use of gibson assembly, a method allowing for the joining of several DNA fragments into one, will be used the following week in attempts to achieve 7kb plasmid isolation without having to restart the process from inoculation onward.

 Introduction This week, we performed a successful inoculation of D. aquaticus from plate to broth. The broth was used later in the week for DNA extraction using the Zyppy Plasmid Miniprep Kit. Procedure The following steps were followed to inoculate D. aquaticus from plate to broth: - A flask of appropriate size should already have media in it and be labeled with initials, date, bacteria name, and type of media. The flask should have been autoclaved and made ready for inoculation. - Gather a bunsen burner, an inoculation loop, and a plate. - Heat the inoculation loop over the bunsen burner flame to sterilize the loop, and let it cool for about 20 seconds. - Open the flask and have the lid in between your fingers underneath the loop. - Swipe the lip of the flask over the bunsen burner flame to sterilize. - Open the plate just enough to collect one colony with the inoculation loop. Close the plate. - Place the loop with the colony into the flask and sweep it around to spread the bac...

Introduction This week in lab, we performed a gram stain on bacteria D.Ficus. Being that it was my first week in the lab, I needed to learn proper labeling and basic techniques used in this lab. The purpose of gram staining is to verify the inoculated colonies have no contaminants. The gram stain allows for differentiation between gram-positive and gram-negative cells. The first two dyes used in a gram stain, violet and iodine, form the CV-I, or crystal violet complex. The crystal violet complex cannot be easily rinsed from gram-positive bacteria because the thick peptidoglycan wall traps it. Gram-negative bacteria, however, have a thin layer of peptidoglycan in their cell wall, which cannot hold the dye. The final dye, safranin, is simply meant to stain the gram-negative cells so they will still show up under the microscope despite having been rinsed of the CV-I complex. Since D.Ficus is a gram-positive bacteria, the expected result is that all of the cells on the slide will be violet...