RNA isolation

 After sending out the prepared samples, we are switching from working with both Deinococcus deserti and D. pimensis to just D. deserti. This week we spent our time planning the next stages of our project as well as running a RNA isolation that was prepped o 8/27 making it a to day sample, and making some media. 


Before the RNA procedure the samples were rehydrated using 500uL rehydration per spot and the plate was then placed on rocker for 15 minutes at 120 rpm to rehydrate. We then used a cell scraper to gather the cells and spin them down to a pellet before stating the procedure. 


RNA:

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

A

62.72.150.97

B

130.71.891.01


While the samples desiccated for 10 days total and ended up slightly dirty, we should be able to perform a clean-up and gel next week. Given the success of plating on the kapton plates we will continue to use them for the main project and it should hopefully increase our yield.

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