Summer
Week of 5/28 - Project Initiation
During the first week of our summer research project, we focused on laying the groundwork for our experimental work. We began by preparing R2A and R2B media and discussing our project timeline and potential research directions. On May 30th, we inoculated our media with Deinococcus sonorensis from our freezeback, carefully incubating the cultures at 30°C.
Week of 6/3 - Experimental Setup
Our plates showed Luteus contamination, prompting us to perform four-way streaking to isolate our cultures. We began developing our project conditions and created Google Drive folders to organize our documentation. On June 4th, we created dedicated Google Drive folders for project documentation and initiated our first desiccation experiment. This involved preparing 10µL dots from 24-hour growth, diluting samples in 500µL media, and inoculating a 24-well plate in triplicate. We also prepared a new plate of D. sonorensis for future experiments. By June 5th, we refined our pre-desiccation sample preparation, adjusting the optical density to 0.84 and performing serial dilutions.
Week of 6/10 - Refinement of Procedures
June 10th we rehydrated our previous samples and conducted further serial dilutions, carefully plating the results in triplicate. On June 11th, we engaged in detailed conversations about DNA and RNA sequencing, with Madison preparing for an upcoming presentation.
Week of 6/17 - Methodology Review
We reviewed our previous desiccation procedures, analyzing both pre- and post-desiccation results. We encountered some challenges, including difficulties with nanodrop readings due to insufficient sample homogenization. Despite these obstacles, we continued to refine our experimental approach, rehydrating previous samples and exploring various sampling techniques.
Week of 6/24 - Experimental Expansion
Our focus during this week intensified on RNA isolation techniques. We conducted 24-hour desiccation experiments, tested various rehydration solutions, and created multiple spot plates to examine sample characteristics. On the 24th we did pre-desiccation procedures and ended with two 24well plates, each plotted in triplicate 10ul dots as well as double pre desiccation dot plates. the next day we rehydrated the samples to test our rehydration solution and did a spot plate. Primer design discussions and online meetings with Chad helped shape our research direction.
Week of 7/1 - RNA Isolation Initial Attempts
By July 1st, we initiated our first significant RNA isolation test, exploring different rehydration times and buffer conditions. Starting w/ 50 uL of rehydration buffer and no rnase inhibitor. Tested time period for rehydration: 15 min (sample C), 22.5 min (sample B), 30 min (sample A). Non desiccated cells were placed in 50 uL of rehydration buffer and samples were taken directly from wells, added to tubes, then spun down. Our initial results showed low RNA yield, prompting a strategic decision to increase our spot size from 20µL to 40µL to improve cell count and RNA extraction. We prepared new 24-well plates with carefully measured D. sonorensis samples, creating quadruplicate spot plates and continuing our RNA isolation attempts. Our July 3rd experiments showed improved results, with RNA isolation yielding more promising outcomes.
Week of 7/8 - Logistical Challenges
Research momentum slowed with team members out of lab. We continued maintaining cultures (inoculating new plates from slants) and preparing media.
Week of 7/15 - Protocol Development
Continued project planning and culture maintenance, inoculating 3 plates and a slant. We conducted pre-desiccation procedures for multiple time points, exploring variations in cultivation and isolation techniques. Our RNA isolation attempts continued, with multiple isolations and cleanup procedures to address consistently low yield.
Week of 7/22 - Continued Experiments
we expanded our research to include 6-day RNA isolation and began preparing for the end-of-summer presentation. The period culminated in a Lab gathering on July 25th, where we all shared our findings and discussed our research trajectory.
Week of 7/29 - Final Experiments
A significant milestone occurred during this final research period: our transition from Deinococcus sonorensis to D. deserti. This shift required substantial protocol modifications, including reducing wash volumes and adjusting desiccation parameters (washes switched from 50 mL to 1mL, no desiccation longer than 4 days). We carefully spotted new sample plates 24 well plate w/ 100 uL of sample to be used for RNA isolation, conducted RNA isolations, and refined our experimental approach. Our control sample on July 31st achieved promising results, with a successful RNA extraction yielding 326.9 ng/µL. On the 1st we again did RNA isolation, 48 hr desiccation D. deserti and 24 hr desiccation deserti on coupons. We rehydrated coupons in 50 mL conical, changing in future to increase overall yield, but of cells were left on the coupon after rehydration
Week of 8/5 - Wrap-up and Reflection
We ran an RNA isolation with the new rehydration method (straight in the well, used cell scrapers as well to get all cells off the plate). The 7 day RNA isolation was ran in tandem, and we upped rehydration from 100 uL to 300 uL. samples were. Rehydrated and rocked for 30 minutes.
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