Reference Gene Project, Start of Fall Semester

 We started lab again this week picking up where we left off after the summer. We have switched species to Deinococcus deserti for the reference gene and have also started working on the new desiccation project as well with the species Deinococcus pimensis. 

We started with new samples of our bacterias as both of these samples were for the second project as opposed to the reference gene project, clearing for contaminations and inoculating in order to go into our pre-desiccation procedure with 24 hours of growth, as well doing the pre-desiccation procedures on a sample of deserti that had grown over the weekend and was intended for RNA isolation following 48 hours of desiccation. The next day 24 hour growth deserti was plated for 24 hours and 7 days of desiccation and 24 hour growth pimensis was plated for 24 hours, 48 hours and 7 days of desiccation . 


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. then repeat
  3. Add second mL of culture+media sample to pack cells
  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 nuclease 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.



The 24 hour pimensis sample didn't dry enough, so they were desiccated an extra 24 hours and processed with the originally intended 48 hour sample. The deserti 48 hour sample was processed the day before. The RNA isolation procedure was as followed:

  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 results of the RNA isolation

deserti 48 hr

77.6 ng/μl2.10  A260/A280

2.17  A2600/A230

pimensis '24' hr

159.1 ng/μl

1.90  A260/A280

0.79  A2600/A230

pimensis 48 hr

165.3 ng/μl

2.01  A260/A280

2.00  A2600/A230



pimensis samples, '24' then 48

48 hr deserti sample



Conclusion

The issues with the pimensis RNA sample originally meant for 24 hours that ended up being processed after 48 hours was most likely due to the issues with drying, and that is was removed for the desiccation environment half way through its total time then replaced. The samples were also all washed and plated on the benchtop this time, rather than the biohood, making it possible for contaminations to be introduced during the process but the other samples processed did not have that issue making it less likely.

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.

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