Week of 9/9/24-9/13/24

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 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. transfer the mixture into a Zymo-spin IICR column in a collection tube in centrifuge for one minute. Discard the flow through
7. add 400μl RNA wash buffer to the column and centrifuge for one minute, then discard the flow through
8. prepare the DNase 1 reaction mix (75μl DNase buffer and 5μl DNase per tube) and add 80μl directly into column matrix and incubate at room temperature (20-30°C) for 15 minutes, then proceed with purification
9. add 400μl RNA prep buffer into the column and centrifuge for one minute. Discard the flow through
10. add 700μl RNA wash buffer into the column and centrifuge for one minute. Discard the flow through
11. add 400μl RNA wash buffer into the column and centrifuge for one minute. Discard the flow through
12. add 400μl RNA wash buffer into the column and centrifuge for two minutes. Discard the flow through
13. transfer the column into a nucleus free tube, and add 50μl DNase/ RNase free water directly to column matrix and centrifuge

Freezeback tubes are pre-prepped tubes with 500uL of 60% glycerol. 1.5mL of liquid culture was added to the tube, then placed in the -80°C freezer for storage and later use

Results & Discussion

Results of the RNA extraction are as follows, with sample 1 being D. deserti, and 2 being D. pimensis

Both samples show low overall yield, but are composed largely (or entirely, according to the reading for D. deserti) of RNA, though both also have contaminants. This lack of yield is most likely from the amount of time between plating and extraction as well as having to go through transit in two directions. It was noted before extraction that the samples were incredibly dry and flaky, with flakes of cells littering the insides of the plates. This was clear for D. pimensis due to its red/pink coloring, but flakes of D. deserti were also assumed to be in a similar state. The contaminants could partially be due to the remaining adhesive on the kapton squares after removal, which would have been allowed to mix with the cells as they rehydrated; it could also simply be from the constant opening and closing of the plates that had to be done to get as many cells as possible back onto the kapton in the first place.

Conclusion

The samples we got back were in, to put it lightly, less than ideal conditions. Given the amount of stress the cells were put under it is of no small significance that we were able to obtain any RNA from them at all. That being said, this puts a pin in this side project entirely for now, aside from running a gel to see if the RNA we extracted was even any good. In the future, we continue our work with Deinococcus deserti. 

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