AMC In Depth

 Intro

After a few days of recuperation time, it was ultimately decided that the characterization of deinococcus sonorensis was effectively over for us, and we would be completely pivoting back onto the AMC project, as well as a more in depth approach into qPCR and investigating reference genes and their stability. 

Results & Discussion

To help further understand the activated methyl cycle, it was drawn out in skeletal structures, as shown above (note: does not contain all components of AMC). As the cell goes through the cycle, pieces of each are taken and substituted and create the substrates present in AMC. While we would have liked to start back up with RNA isolation and qPCR procedures for this project, there were issues with both e. coli and D. caeni samples that prvented us from doing so. 

With the addition on the qPCR project we are now adding to the workload and having to better understand the process of qPCR and also what reference genes would best benefit us and the lab. Given a list of reference genes, we were able to narrow down the full list down to era, fabD, ftsZ, groEL, rho, ropB and rpoD. This was done by selecting only those with a standard deviation of +/- .7 or lower. However era and ftsZ do not have the standard deviation data so we may end up cutting those. We also looked into each of the genes and where they come from/what they are associated with in order to get a better idea of each gene.

Conclusion
We will be testing each reference gene listed above with qPCR and also continuing the RNA isolation of e. coli and D. caeni into the summer and hopefully be done with these projects before the conclusion of the summer semester. 

Comments

Post a Comment

Popular posts from this blog

UV Exposure

  Intro Much of this week was dedicated to filling out our poster. What wasn't spent on poster work was spent on testing UV radiation tolerance again. The MRVP tests were done again and were again decided to be inconclusive. Method Method for UV exposure is exactly the same as last week, but R2A was exposed at levels of 1000, 1250, 1500, 2000, & 3000. TGY was exposed to 3000, 5000, 6000, 7000, 8000, & 9000. Results & Discussion Again, there was absolutely no growth on any of the R2A plates. The threshold is cleary far lower than we expected due to the media of R2A while, again on TGY, there was growth from the 3000 & 5000 plates. Conclusion Really all we have left moving forward is little details and pictures for the poster. All of the tests have been conducted at least once, but a few of them need to be redone still to see if we can verify a true positive/negative result. 
Read more

Week of 10/14/24-10/18/24

Image
Intro This week involved another round of RNA isolation, followed by purification and then an RNA gel and fluorometer check to ensure quality of the RNA before proceeding.  Methods Before RNA isolation, cells were rehydrated with 650uL of rehydration buffer per 100uL spot, then allowed to sit on the rocker for 15minutes at 250rpm to fully rehydrate. Kapton squares were then scraped to ensure removal of the cells, then pipetted into 1.5mL eppendorf tubes. 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 into a Zymo-Spin IICG column in a collection tube and centrifuge for one minute. Save the flow through 5. Add an equal volume of ethanol (95-100%) and mix well 6. tra...
Read more

Ref Gene Progress

Image
Intro     As we return to lab for the fall, we pick up exactly where we left off from the summer. With the reference gene project we've switched species to Deinococcus deserti, and with that Alex has taken time in the week inbetween to become familiarized with it. As we are officially working on the new desiccation project as well, we have been given a second species to continue our work with: Deinococcus pimensis. Over the course of the week we plated samples of both species for 24 hour, 48 hour, and 7 day desiccation.  Methods On Monday 8/19/24, a recently grown sample of deserti was gram stained to ensure no contamination before being used for inoculation. We found gram negative rods with no contaminants, so the plate was used for inoculation. A different newer plate that had left to grow over the weekend was used for starting 48hrs of desiccation to extract RNA. The 24hr and 7 day deserti, as well as the 24hr, 48hr, and 7 day pimensis were all plated the followin...
Read more