Mar 27, 2025
Total RNA extaction of Staphylococcus aureus
This protocol is a draft, published without a DOI.
- Kim Jung-Min1
- 1Asan Medical Center, Seoul, Korea
Protocol Citation: Kim Jung-Min 2025. Total RNA extaction of Staphylococcus aureus. protocols.io https://protocols.io/view/total-rna-extaction-of-staphylococcus-aureus-d6p59dq6
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: March 27, 2025
Last Modified: March 27, 2025
Protocol Integer ID: 125405
Abstract
Because of the thick and complex cell wall of S. aureus, special enzymes are required for RNA extraction, but when Trizol is used, sufficient RNA can be extracted without expensive enzymes and long reaction times.
Materials
Trizol (Thermo, #15596026) or Qiazol (Qiagen, #79306)
Chloroform
70% Ethanol
RNA purify kit with spin column
RNA extraction of Staphylococcus aureus
RNA extraction of Staphylococcus aureus
Culture S. aureus in TSB media until appropriate time.
Harvest S. aureus pellet by centrifuge for 15 min at 7,000 rpm.
Disrupt the cells.
Add TRIzol 400 μl to pelltet and lysis with pipetting, and incubate for 10 min at RT
(When the pellet mass is about 50 μl, add 400 μl of TRIzol).
Add chloroform 80 ul and, tap mix and incubate 2 min at RT.
Centrifuge for 15 min at 4℃ and 12,000 rpm .
At this time, prepare the DNase I solution (add 10 μl DNase I stock to 70 ul Buffer RDD and stor on ice).
Transfer upper clear phase to new e-tube.
Add equal volume of 70% Ethanol and vortex mix.
Transfer 700 μl of sample to spin column
(Qiagen RNeasy or any RNA purify kit).
Centrifuge for 15 s at ≥ 10,000 rpm and discard the flow-through
(Afterwards, this process is called **spin down**).
Add 350 μl RW1 (WashⅠ) to spin column and spin down.
Add 80 μl DNase I mix directly to the spin membrane, and incubate for 10 min at RT.
Add 350 μl RW1 to spin column and spin down.
Add 500 ul RPE (WashⅡ) and spin down.
Centifuge at full speed for 2 min.
Plas the spin column in a new e-tube and add 50 μl RNAse-free water directly to the spin column membrane.
Incubate for 1 min and centrigue 1 min at 12,000 rpm.
Store eluting RNA at -80℃ until use.