More Ref Gene

 Intro

    This week we completed the 7 day desiccation and RNA isolation of Deinococcus pimensis and Deinococcus deserti. Though there were some slight mishaps, the overall results were promising.

Methods

The procedure for RNA isolation is the same as listed previously

  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

The procedure for packing and plating cells for desiccation is as follows:

  1. suspend cultures in 10mL of nuclease free water
  2. take OD reading and normalize to 1
  3. spin down cultures (5000 RCF, 4°, 5 min always)
  4. remove supernatant, resuspend in 4mL water
  5. spin down cultures, remove supernatant, resuspend in 4mL water
  6. spin down cultures, remove supernatant, resuspend in 2mL water for plating
  7. take final OD value
  8. plate 12 100uL samples onto kapton, each in a well of a 6 well plate
During spinning, an extra 200uL of deserti was added to its sample in order to match weight of pimensis tube for centrifuge.

Results and Discussion

    Unfortunately, as 7 day samples were starting to be worked on for RNA isolation, there was an issue with mislabeling, making the deserti sample unusable. The pimensis sample, however was fine and showed good results (190.7ng/uL, 2.01, 2.17). However, there was a sample from the previous week meant to be a 24hr deserti sample that did not get used completely, so there were spots leftover with cells that had been sitting for a full week. When they were used on 8/28/24, they gave the following results:

The 1st set of results comes from a sample that was plated 24 hrs prior, while the 2nd is the above mentioned 8 day sample. Both of these were plated directly into the wells of the well plate, not onto kapton as is procedure for the desiccation set. Generally, both show lower results for yield than are typical for the cells plated onto kapton. While not as great as the previous week's samples, these are still decent as long as we run a cleanup before using them. More importantly, they show that the RNA can and will survive over an 8 day period.

Conclusion

    With all of this side project done for now except for little details, we are able to pivot and focus fully on the ref gene project moving forward. Being optimistic, we should be able to get to the RNA-seq portion within the next couple weeks. 


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