S-STEM Scholar Alex Hugenberg-Cox Blog

 We started the week off strong with a pre-desiccation procedure, then assisting Evan with a procedure, and finally going over our sequencing protocol.  We did pre-desiccation on our A B and C flasks grown last week, following the usual cell packing procedure and then moved them to the egg the following day. Friday they were removed from the vacuum as it was unable to hold pressure that long but we were unable to perform RNA iso, so they dehydrated over the weekend.  1. Normalize 3ml of culture to an OD between 0.95-1.00 2. Spin down 1ml of media, remove supernatant and resuspend pellet in nuclease free water 3. Resuspend pellet, then centrifuge and remove supernatant 4. Add the second ml of culture, repeat washing steps 5. Add the third ml of culture, repeat washing steps 6. Plate 100ul dots into 1 inch kapton squares in a 6 well plate in triplicate We were also able to help Evan with a twitch motility procedure, preparing the specialized plates with the unique molds and...

 We started the week with RNA isolation, then did a clean up kit followed by a gel and fluorometer analysis. We did the normal RNA isolation procedure, then after analyzing the results did a clean up the next day. We did a Midori green RNA gel ( 1%, 50ul gel with 5ul of Midori Green)  from those results, and analyzed the sample quality the next day. RNA: 1. Resuspend a fresh or frozen pellet in 800μl RNA lysis buffer and transfer the mixture to a ZR bead bashing lysis tube 2. Secure the tube on a bead beater with a 2ml tube hold assembly and process. Repeat 5 cycles of 1 minute on and 2 minutes on ice 3. Centrifuge the tube for one minute to pellet debris 4. Transfer up to 400μl of the cleared supernatant into a Zymo-Spin IICG column in a collection tube and centrifuge for one minute. Retain the flow through 5. Add an equal volume of ethanol (95-100%) and mix thoroughly 6. Transfer the mixture into a Zymo-spin IICR column in a collection tube and centrifuge for one minute. Dis...

 We started the week inoculating 3 plates and 3 flasks from freeze back, then later in the week did a pre-desiccation procedure and discussed more about what the RNA sequencing would look like.  For pre desiccation we did the usual procedure, let it dehydrate for two days then placed it in the Egg, which is our new desiccator. Unlike the old glass one, this one is sealed and vacuumed making it true desiccation. Dehydration is a process of removing water or moisture from a substance, typically through heat, air circulation, or other drying methods. In dehydration, some water remains within the material, and the substance typically retains some degree of moisture. True desiccation, on the other hand, is a more extreme form of water removal that aims to completely eliminate all moisture from a substance.  A vacuum is not absolutely necessary for true desiccation, but it can be an extremely effective method for removing moisture. In the desiccation process, a vacuum proves va...

 Like usual we started this week with RNA extraction, as well as inoculating for and planning the upcoming weeks. We made new media and inoculated from freeze back to prep for pre-desiccation next week. Our RNA sample was from a four day desiccation period, and we went from 500ul to 250ul of rehydration buffer to make up for a limited supply of the buffer this week. The samples also sat in the rocker for 10 extra minutes to properly rehydrate with the limited amount of buffer. RNA Isolation: 1. Resuspend a fresh or frozen pellet in 800μl RNA lysis buffer and transfer the mixture to a ZR bead bashing lysis tube 2. Secure the tube on a bead beater with a 2ml tube hold assembly and process. Repeat 2 cycles of 1 minute on and 2 minutes on ice 3. Centrifuge the tube for one minute to pellet debris 4. Transfer up to 400μl of the cleared supernatant into a Zymo-Spin IICG column in a collection tube and centrifuge for one minute. Retain the flow through 5. Add an equal volume of ethanol (9...

 Most of this week was spent on the returned samples of D. deserti and D. pimensis . When going to rehydrate the samples it was noticed that while all the samples were extremely dry, the pimensis samples were flaky and had fallen off the kapton in some cases. We also ran into issues when looking at the tape used to stick the kapton down, as we didn't want to introduce any more organic contaminates as that's already something we struggle with. During a discussion a number of options were discussed such as curling up the kapton sides to create a bowl of sorts, rehydrating the samples face down and using a much lower volume of rehydration buffer. In the end we removed the kapton squares from the original plates, removed the tape best as possible and placed then in a new six well plate for rehydration.  The samples were rehydrated with 500ul of rehydration buffer per well, placed onto the rocker for 15 minutes. The kaptons were then scraped to make sure all cells were removed and ...

 After sending out the prepared samples, we are switching from working with both  Deinococcus deserti  and  D. pimensis to just  D. deserti . This week we spent our time planning the next stages of our project as well as running a RNA isolation that was prepped o 8/27 making it a to day sample, and making some media.  Before the RNA procedure the samples were rehydrated using  500uL rehydration per spot and the  plate was then placed on rocker for 15 minutes at 120 rpm to rehydrate. We then used a cell scraper to gather the cells and spin them down to a pellet before stating the procedure.  RNA: 1. Resuspend a fresh or frozen pellet in 800μl RNA lysis buffer and transfer the mixture to a ZR bead bashing lysis tube 2. Secure the tube on a bead beater with a 2ml tube hold assembly and process. Repeat 2 cycles of 1 minute on and 2 minutes on ice 3. Centrifuge the tube for one minute to pellet debris 4. Transfer up to 400μl of the cleared superna...

This week we completed the 7-day desiccation and RNA isolation process for Deinococcus pimensis and Deinococcus deserti, as well as completed our side projects pre-desiccation protocol. Despite a few minor setbacks, the experimental outcomes showed promising potential. Our findings demonstrate the RNA's capacity to survive for up to 8 days. With this side project mostly completed, the team can now redirect full attention to the reference gene project. Optimistically, the RNA-seq phase is anticipated to commence within the next couple of weeks. Protocol: The RNA isolation procedure remains consistent with previous protocols, 1. Resuspend a fresh or frozen pellet in 800μl RNA lysis buffer and transfer the mixture to a ZR bead bashing lysis tube 2. Secure the tube on a bead beater with a 2ml tube hold assembly and process. Repeat 5 cycles of 1 minute on and 2 minutes on ice 3. Centrifuge the tube for one minute to pellet debris 4. Transfer up to 400μl of the cleared supernatant into a...