Ref Gene Progress

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 following day (8/20/24).

Pre-desiccation procedures were as follows:

1. in 1mL of media, suspend culture equal to OD=1, (3x)
2. centrifuge to pellet, remove supernatant, resuspend in 1mL of nuclease free water, pipette up and down (10x) to mix. Repeat.
3. centrifuge to pellet, remove supernatant, add the other tube of culture+media to pack
4. repeat step 2 to wash (2x)
5. repeat step 3 for remaining tube, then repeat step 2 to wash
6. remove supernatant, resuspend in 500 uL of muclease free water

All samples were plated onto kapton coupons placed in 6 well plates. To desiccate, 100uL of sample was plated directly onto kapton coupons and left to dry.

RNA isolation was completed for the 24 and 48hr samples of both species 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 and Discussion

    As it's been on the humid side this week, there was a slight issue with getting the cells to dry down in time for extraction, specifically with the 24hr samples. As such, all results are for 48 hr samples instead.

Results above are for the 48hr deserti sample

Results above are for the "24"hr and 48hr pimensis samples, in that order

    (note: "24" hr as is was plated to be a 24 hr sample but took 48 hrs to dry but 48hr and 48hr is confusing)

All samples were washed and plated on benchtop, rather than in the fume hood this time around. It is possible that the contaminants in the result above are simply from poor aseptic technique, or more likely, a momentary lapse. As the other samples were plated the same day and are clean, it is likely as simple as not paying attention for one moment which unfortunately compromised the integrity of our sample.

For all of the samples, protocol called for them to be centrifuged for 6 minutes per round, as indicated in the procedure. With 5 samples, each spread out across 3 tubes, that means I had to complete 6 rounds of 6 minutes in the centrifuge, making the packing a huge time killer. Doing all 5 samples in one sitting, as well as having to mix each sample by hand before centrifuging to wash, this procedure took about 4 hours to complete on my own. Whether that be due to a lack of speed or not, this was still bound to take at least 36 minutes on washes alone. This procedure can easily be cut down to reduce the time strain by cutting the wash time by at least in half, though we have decided to reduce it down to 1 minute per cycle.

Conclusion

    With the results for our 7 day samples not being available until 8/27, it's a it unclear where next week will leave us. If all goes well, both samples will be clean and come out with high yield. From there, it'll likely be us moving back towards our personal project, the reference gene project, and marching closer to RNA-seq.