Week of 9/2/24-9/6/24

 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. 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

RNA isolation procedure was run on samples left to desiccate for 10 days total, and were labeled A&B, respectively. Sample A, when measured post procedure, gave RNA results of 62.7ng/uL, a 260/280 value of 2.15, and a 260/230 value of 0.97/ Sample B gave readings of 130.7ng/uL, 1.89, and 1.01. Based on these values, it is likely a clean-up procedure will be done in the following week, but couldn't be done this week due to time constraints. Notably, sample B was still cloudy post bead beating step in the procedure, but still came out with higher yield and less organic contaminants.

Conclusion

Despite the samples coming out relatively dirty, the 10 day desiccation was largely a success. With >60ng/uL in yield of RNA, we should be able to run more gels for quality before moving forward in the direction of sequencing, so long as we continue to hone in on the procedure for the sake of replicability. During the discussion of this week's plans, it was also decided that moving forward all samples would be plated onto kapton squares to (hopefully) increase yield consistently.

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