S-STEM Scholar Madison Jackson's Blog

Intro Once again, the week was dedicated to desiccation and RNA extraction procedures. Notably, there were interesting results from some of the gram stains, and it was decided to make new freezebacks because of this.  Methods Samples A, B, & C were OD'ed and normalized to a value of 1.0 (+/- .1), then cells were packed by centrifuging, washing, then adding 1mL of OD'ed sample from a separate eppendorf tube. Samples were plated onto kapton squares in a 6 well plate, with spots containing 100uL of sample each. Samples were also placed into *new* desiccator to sit over the weekend for a total of 2 days of drying time and 2 days in the desiccator. Results & Discussion Cells that were gram stained this week looked like this: The gram negative color is characteristic of D. deserti , but the cells are usually rod-shaped. All of the gram stains done showed the same gram negative color but cocci shape. Despite this, it was decided to move forward because occasionally cells that ...

Intro More time this week was again dedicated to future planning, and more desiccation procedures. Samples for RNA extraction were from a four day desiccation period, having been left in the incubator since the previous Wednesday 9/15/24. New samples were inoculated on Friday, 9/20/24, to grow over the weekend and to be later used for more concrete desiccation procedures. Methods Notes before RNA extraction: limited amounts of rehydration buffer available, switched from 500uL per spot to 250uL per spot to compensate samples allowed to sit for an extra 10 minutes on rocker to rehydrate with the limited amount of buffer RNA isolation procedure: 1. Resuspend fresh or frozen pellet in 800μl RNA lysis buffer and transfer the mixture to a ZR bashingbead lysis tube 2. Secure the tube on a bead beater fitted with a 2ml tube hold assembly and process. 1 min on/2 min ice repeated 5 times 3. centrifuge the tube for one minute to pellet debris 4. transfer up to 400μl of the cleared supernatant int...

Intro Samples that were sent out 8/30/24 for the kapton side project with both D. deserti and D. pimensis were returned this week, and that became the bulk of the work this week. Aside from that, time was spent inoculating samples, gram staining them, and then creating freezebacks of those samples of D. deserti for future use, as well as assisting other groups. Methods Before RNA isolation: plates containing cells and kapton were scraped to try and get all cells onto the kapton kapton squares with cells were removed from original 6 well plate and moved into new 6 well plate samples were rehydrated as normal with 500uL of rehydration buffer per 100uL spot, with samples from each plate being combined into one to increase overall yield (2 plates D. deserti , 2 D. pimensis , 1 plate for DNA extraction and 1 for RNA) once rehydration buffer was added, samples went onto rocker for 15 minutes at 120 rpm after rehydration, kapton was scraped down to ensure all cells were removed and added into...

  Intro After prepping and sending out samples for the previous week, we are pivoting to working entirely with Deinococcus deserti and D. pimensis is falling to the wayside. Time this week was dedicated to planning for the upcoming weeks and preparing. A round of RNA isolation was run on 10 day samples plated on 8/27/24 as well. Methods TSB10 was made in batches of 250mL and 125 mL, as well as 250mL of TSB10 with agar for plates. Media was made using the recipe on the bottle followed by a tenfold dilution. RNA procedures were with the following notes: Pre procedure, spot plates were rehydrated using 500uL rehydration per spot plate was then placed on rocker for 15 minutes at 120 rpm to rehydrate RNA isolation for zymo kit is as follows: 1. Resuspend fresh or frozen pellet in 800μl RNA lysis buffer and transfer the mixture to a ZR bashingbead lysis tube 2. Secure the tube on a bead beater fitted with a 2ml tube hold assembly and process. 1 min on/2 min ice repeated 5 times 3. centri...

  Intro     This week we completed the 7 day desiccation and RNA isolation of Deinococcus pimensis and Deinococcus deserti. Though there were some slight mishaps, the overall results were promising. Methods The procedure for RNA isolation is the same as listed previously Resuspend fresh or frozen pellet in 800 μl RNA lysis buffer and transfer the mixture to a ZR bashingbead lysis tube Secure the tube on a bead beater fitted with a 2ml tube hold assembly and process. 1 min on/2 min ice repeated 5 times centrifuge the tube for one minute to pellet debris transfer up to 40 0 μl of the cleared supernatant into a Zymo-Spin IICG column in a collection tube and centrifuge for one minute. Save the flow through Add an equal volume of ethanol (95-100%) and mix well transfer the mixture into a Zymo-spin IICR column in a collection tub e in centrifuge for one minute. Discard the flow through add 40 0 μl RNA wash buffer to the column and centrifuge for one minute, then discard th...