Side Project RNA Isolation

 Most of this week was spent on the returned samples of D. deserti and D. pimensis. When going to rehydrate the samples it was noticed that while all the samples were extremely dry, the pimensis samples were flaky and had fallen off the kapton in some cases. We also ran into issues when looking at the tape used to stick the kapton down, as we didn't want to introduce any more organic contaminates as that's already something we struggle with. During a discussion a number of options were discussed such as curling up the kapton sides to create a bowl of sorts, rehydrating the samples face down and using a much lower volume of rehydration buffer. In the end we removed the kapton squares from the original plates, removed the tape best as possible and placed then in a new six well plate for rehydration. 


The samples were rehydrated with 500ul of rehydration buffer per well, placed onto the rocker for 15 minutes. The kaptons were then scraped to make sure all cells were removed and gathered, and 3 wells were combined to one sample to increase yield. In the end we had 4 samples, 2 deserti  and 2 pimensis, each one having one sample meant for DNA and one for RNA. 


RNA Isolation:

1. Resuspend a fresh or frozen pellet in 800μl RNA lysis buffer and transfer the mixture to a ZR bead bashing lysis tube

2. Secure the tube on a bead beater with a 2ml tube hold assembly and process. Repeat 2 cycles of 1 minute on and 2 minutes on ice

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. Retain the flow through

5. Add an equal volume of ethanol (95-100%) and mix thoroughly

6. Transfer the mixture into a Zymo-spin IICR column in a collection tube and centrifuge for one minute. Discard the flow through

7. Add 400μl RNA wash buffer to the column, centrifuge for one minute, and discard the flow through

8. Prepare the DNase 1 reaction mix (75μl DNase buffer and 5μl DNase per tube), add 80μl directly to the column matrix, and incubate at room temperature (20-30°C) for 15 minutes before continuing purification

9. Add 400μl RNA prep buffer to the column and centrifuge for one minute. Discard the flow through

10. Add 700μl RNA wash buffer to the column and centrifuge for one minute. Discard the flow through

11. Add 400μl RNA wash buffer to the column and centrifuge for one minute. Discard the flow through

12. Add 400μl RNA wash buffer to the column and centrifuge for two minutes. Discard the flow through

13. Transfer the column to a nuclease-free tube, add 50μl DNase/RNase-free water directly to the column matrix, and centrifuge



ng/ul

a260/280

260/230

Deserti

44.02.061.34

Pimensis

21.81.940.68


While both samples had low overall yield, they were composed of largely RNA although they many organic contaminates. The samples were incredibly dry and flaky, and the were exposed without lids before they were shipped back to us, both of which most likely contributed to the numbers seen. It could also be due to the remaining adhesive from the tape, as well as messing with the flakes to try and collect them all. 

Overall the samples were in extremely poor condition and had been put under immense stress, making it lucky we were able to receive as good of numbers as we did. Over all this is it for this side project for now, but we did take the time this week to make freezebacks incase we end up returning to the project.

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