S-STEM Scholar Madison Jackson's Blog

Introduction        The main focus of this week was completing set-up for quantitative polymerase chain reaction, or qPCR. This was a shortened week with the Monday holiday, so a large portion of the week was dedicated to setting up and then running qPCR, with a bit of time being set aside to work on tests for the Sonorensis project, specifically sugar fermentation and more experimentation on TGY vs. R2A. At the beginning of the week we had a discussion with Chad about microscopy, though for those on the AMC project it was shorter than for those on sonorensis. Methods     Shown above are the calculations done for qPCR setup For each of the sugar fermentation tests, they were inoculated via our usual method: inoculation loop is heated loop is briefly cooled, then used to collect bacteria to be added to other medium (sugar fermentation tube in this case) collected bacteria is added to new media (sugar fermentation tube) then allowed to grow Tubes were inocula...

Introduction         As mentioned at the end of last week's post, the main focus of this week was completeing E. coli RNA isolation clean enough to be able to continue onward with qPCR. Seeing as Alex needed to be present for the RNA isolation and is busy running multiple projects, this wasn't able to be done until the end of the week. However, a good portion of time was dedicated to Alex teaching Carime and myself about the activated methyl cycle and the premise of our project, which will be discussed in detail at the end of this post. Methods      The procedure for RNA isolation is as follows: 1. Resuspend fresh or frozen pellet in 800uL RNA lysis buffer and transfer misxture into ZR BAshingBead Lysis tube (0.1 and 0.5mm) 2. Secure tube in bead beater fitted with 2mL tube hold assembly and process 3. Centrifuge tube for 1 minute to pellet debris (1 min. on, 2 min. off) 4. Transfer up to 400uL of cleared supernatant into a Zymo-Spin IIICG Column...

February 11, 2024 Introduction The bulk of this week was dedicated to the process of ethanol precipitation. Based on the 260/230 numbers from the nanodrop last week, it was clear that a contamination clean up was necessary, which in this case we used the ethanol precipitation procedure. Also done this week was the making of media for the sonorensis project, along with inoculation on each type of plate at the end of the week. Methods The procedure for ethanol precipitation is as follows: 1. Add .1 vols. 3M sodium acetate 2.5-3 vols ICE COLD 100% ethanol 2 uL glyco blue Vortex to mix thoroughly 2. Precipitate (ppt.) at -20℃ for 1 hour or overnight or at -80℃ for 1 hour (overnight will give more ppt. If RNA amount is low) 3. Centrifuge at full speed (13000 rpm) and 4℃ for 30 minutes 4. Wash pellet twice with .5ml ICE COLD 75% ethanol, spinning at 4℃ for 10 minutes each time 5. Take ethanol out, spin quickly (10s top speed) to remove as much trace amounts of ethanol as you can 6. Ai...

  February 4, 2024 Introduction This week included RNA extraction from E. coli with the intention of working towards qPCR, but also to test the kit for organic contaminants. Also this week was an introduction into gram staining, which is used specifically to verify samples and to check for contamination. Finally this week was inoculation, the process of implanting a microbe or other organism onto a plate for further testing later.  Methods For RNA extraction and purification, the steps are as follows: resuspend fresh or frozen cell pellet in 800uL RNA Lysis buffer and transfer the mixture into ZR BashingBead Lysis Tube. Secure the tube in a bead beater fitted with 2mL tube hold assembly and process. Centrifuge the tubes for 1 minute to pellet debris, then let sit in an ice bath for 2 minutes. Repeat ten times, then transfer up to 400uL of cleared supernatant into a zymospin IIICG column in a collection tube and centrifuge, saving the flow-through. Add an equal volume ethan...

  January 28, 2024 Introduction This week was an introduction to the TRAIN program for new and returning members. Lani gave a presentation about the program that included general safety information, a brief introduction into the bacteria Deinococcus, and the general expectations of the program. For example, these blog posts are required at the end of the week every Sunday detailing the work for the week and the methods and results from said work. Lani also supplied a few resources that should come in handy further into the semester, such as Google Scholar, PubMed, and BacDive. These resources should be used to gain information in order to know more about bacteria we work with here in the lab, as well as to help find information in the future for projects. Also this week was a brief introduction into making media and using the nanodrop. Methods The general process to making media is to follow the recipe and dilute. This week, we made TGY (tryptone, glucose, yeast) by diluting the ...