Nov 23, 2020
RNA Direct Lysis Method
This protocol is a draft, published without a DOI.
- 1In-house protocol
Protocol Citation: Elizabeth Fozo 2020. RNA Direct Lysis Method. protocols.io https://protocols.io/view/rna-direct-lysis-method-bpwrmpd6
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: November 19, 2020
Last Modified: November 23, 2020
Protocol Integer ID: 44721
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Abstract
RNA Direct Lysis method
(from Masse and Gottesman. 2003 Genes and Development. 17:2374-2383) (notes and italics are from E. Fozo by Selene Hess)
Guidelines
Direct Lysis solution:
- 320 mM NaAcetate
- 8% SDS
- 16 mM EDTA
N.B. Direct lysis solution will not stay in solution at RT. Prior to use, must get into solution by placing at 37 °C.
Note
The recipe must be altered for use from minimal media.
Steps
Steps
Grow cells to desired OD.
At desired OD, remove 750 ml of cells to a tube at 65°C containing 500 ml acid:phenol-chloroform with 100 ml of direct lysis solution (SET-UP at least 25 minutes prior to harvesting to ensure the components are at 65°C). Vortex to mix, and place the tube back at 65°C for 10-15 minutes.
Note
Can store at -80 indefinitely. Once move on, can’t stop till step 7, ethanol.
Spin 13000 r.p.m., room temperature for 5-10 minutes.
Note
(15-20 minutes if from -80 storage)
Transfer supernatant to a new tube containing 500 ml of 65°C acid phenol: chloroform. Spin as in step 3 (RT).
Transfer supernatant and extract with 400 ml “regular” phenol: chloroform. Gentle vortex. Spin as in step 3 (RT). May need to do additional extractions at this point.
Transfer supernatant to a new tube and extract with 400 ml chloroform. Spin as in step 3 (RT).
Note
N.B. Liz and Aixia are the only ones who do this step, so it is clearly optional!
In interface is thick, white goo.
Transfer supernatant and add 700 ml 99% ethanol (95% is fine). Place in -80 C for 20 minutes (or indefinitely).
Spin 13000 r.p.m. 4°C, 30 minutes. Wash with 400uL 70% ethanol. Let pellet air dry.
Resuspend in RNase free water (20-25uL).
Let sit for 10 minutes at RT and finish resuspending by pipetting up and down/flicking tube.