> For the complete documentation index, see [llms.txt](https://salis-lab.gitbook.io/protocol-book/llms.txt). Markdown versions of documentation pages are available by appending `.md` to page URLs; this page is available as [Markdown](https://salis-lab.gitbook.io/protocol-book/test/rna/rna-extraction.md).

# Spin Column-Based Extraction

`PROTOCOL DUMP PLEASE FORMAT`

Things to check before starting the protocol

* Make 1 mg/mL lysozyme in RNase-free 1x TE buffer \[prepare 150 µL per RNA sample]
* Add 10 µL β-Mercaptoethanol (βME) to 1 mL Lysis Solution (Norgen Kit) in an RNase-free eppendorf tube and mix by quick vortexing \[prepare 400 µL per RNA sample required]
* Thaw an aliquot of yeast tRNA (25 µg/µL stock concentration) on ice

Steps to minimize RNAse contamination

* Cover hands, arms, head, and body with sterile protective gear (RNAses are in your saliva, your tears, and on your body surface).
* Wipe down all bench surfaces with RNAse Away
* Use only sterile, disposable plastic tubes, tips, and serological pipettes. It is important to keep a box set of tips for RNA use only -- do not use these tips for any other labwork, e.g. minipreps.

Protocol Steps

1. Spin down at room temperature 5 mL of bacterial culture at OD600 0.2 (log phase) at max rpm (4,750 rcf), and discard the supernatant \[try to withdraw as much supernatant as possible without disturbing the bacterial pellet]
2. Resuspend the pellet in 100 µL of the lysozyme solution (in TE) by pipetting up and down and/or vortexing and transfer to an RNase-free eppendorf tube. Incubate the resuspension at room temperature for 5 minutes \[if the room temperature is low, incubate at 25 °C]
3. Add 300 µL of the βME containing Lysis Solution (Norgen Kit) and mix by vigorous vortexing for 10-15 seconds
4. Add 200 µL of ethanol (98-100%) to the lysate and mix by vigorous vortexing for 10-15 seconds \[use the EtOH bottle from the designated "RNA ONLY" drawer]
5. Assemble the RNA purification column and the collection tube (Norgen Kit) and add 600 µL of the lysate from Step 4 above to it. Spin at 14,000 rcf for 1 min at room temperature \[if all the lysate does not go through the column, spin for another 1 min]
6. Discard the flow-through and reassemble the column
7. Add 400 µL of the Wash Solution (Norgen Kit) to the column and spin at 14,000 rcf for 1 min at room temperature \[the Wash Solution should have been prepared beforehand by adding RNase-free ethanol to the Norgen supplied bottle]
8. Discard the flow-through and reassemble the column
9. Repeat Steps 7 & 8 two more times (total of 3 washes)
10. Spin the column at 14,000 rcf for 2 min at room temperature in order to dry the column resin
11. Discard the collection tube and place the column into a fresh RNase-free eppendorf tube \[use cold eppendorf tubes]
12. Add 50 µL of the Elution Solution (Norgen Kit) to the column and spin at room temperature at 200 rcf for 2 min, followed by 14,000 rcf for 1 min \[place the RNA on ice-water immediately and keep cold all the time]
13. For greater recovery, place the column back into the same eppendorf tube and repeat the elution with another 50 µL of the Elution Solution
14. Take out a 3 µL aliquot in a separate RNase-free eppendorf tube for later analysis
15. To the rest of the 95 µL RNA, add 9.5 µL of the 10x TURBO DNase Buffer and 2 µL TURBO DNase (TURBO DNA-free™ Kit) and mix gently by flicking the tube
16. Incubate at 37 °C for 30 min
17. Add 10.7 µL DNase Inactivation Reagent (TURBO DNA-free™ Kit) and mix gently by flicking the tube \[before pipetting the DNase Inactivation Reagent, flick the tube a couple of times to ensure that the beads are uniformly dispersed]
18. Incubate at room temperature for 5-6 min while mixing gently by flicking the tube every 2 min
19. Spin at room temperature at 10,000 rcf for 1.5 min and transfer 90 µL of the supernatant to a fresh RNAse-free eppendorf tube \[use cold eppendorf tubes; keep cold all the time]
20. Take out a 3 µL aliquot in a separate RNase-free eppendorf tube for later analysis \[use cold eppendorf tubes; keep cold all the time]
21. Use the nanodrop to determine RNA concentration using the aliquots taken earlier in Steps 14 and 20 (pre- and post- DNAse treatment)
22. Using the Elution Solution (Norgen Kit) dilute all the post-DNase RNA samples to the same concentration 50-150 ng/ µL
23. Run 1 µL each of the RNA samples from Steps 14 & 20 above (pre- and post- DNAse treatment) on a 1% agarose gel to check for RNA integrity.

See [ZR Soil/Fecal RNA MicroPrep (Zymo Research)](http://www.zymoresearch.com/rna/rna-isolation/soil-fecal-plant-rna/zr-soil-fecal-rna-microprep)to see shows bacterial rRNA (23S and 16S)

![](https://lh6.googleusercontent.com/EkuOhkphL6WlCx1cGxDrq1JvoERWL1yVCB_g5ZnTs_mxvoxmtin5PQX5gyL9OlksWHUi3bfYvda369tHjwQQdEFq8FxGbWJDKTyX3qxurCXDgB1lh5SbA47Vw6iKGCjhDg6Vs9yy)

-Two sharp bands at \~1 kb confirm RNA integrity;

* fuzzy/ smeary bands indicate degradation
* Aliquot 20 µL each of the RNA samples from Step 22 into four fresh RNase-free tubes a-d
* Add 0.2 µL of yeast tRNA (25 µg/µL stock concentration) to two aliquots (a & b) from Step 24 above \[end concentration 250 ng/µL tRNA]
* Freeze down all the five tubes at -80 °C: the original tube from Step 22 and tubes a-d from Steps 24\
  &\
  25\.