Week of 9/16/24-9/20/24

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 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

Results & Discussion

Results from the RNA isolation procedure are as follows:

Sample B was lacking in yield, all three samples definitely had the RNA from the procedure, but all three were in varying degrees of cleanliness.  As we have been working on solidifying our procedure and we feel confident in our methodology, we have decided to move towards definitively obtaining samples to be able to sequence. With this in mind, we are not moving forward with a clean-up on these samples and instead to an entirely new set with the following game plan:

Conclusion

With new media made and fresh flasks inoculated from our freezebacks, the plan is to move forward next week with pre-desiccation procedures on Wednesday, as well as creating CFU plates for colony counting and dilutions. Much of the time will be spent letting cells grow, so it is likely that a good deal of time will be spent helping other groups with their projects.

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