Nov 22, 2020
Version 1
DNA extraction for human microbe samples. V.1
Peer-reviewed method
- 1BGI-Shenzhen, Shenzhen 518083, China.
- GigaScience Press
- BGI
External link: https://doi.org/10.46471/gigabyte.9
Protocol Citation: Lilan Hao 2020. DNA extraction for human microbe samples.. protocols.io https://dx.doi.org/10.17504/protocols.io.bcmriu56
Manuscript citation:
Chen C, Hao L, Wei W, Li F, Song L, Zhang X, Dai J, Jie Z, Li J, Song X, Wang Z, Zhang Z, Zeng L, Du H, Tang H, Zhang T, Yang H, Wang J, Brix S, Kristiansen K, Xu X, Wu R, Jia H, The female urinary microbiota in relation to the reproductive tract microbiota. GigaByte. 2020.
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: February 17, 2020
Last Modified: November 24, 2020
Protocol Integer ID: 33169
Abstract
This protocol is used to clarity the process of total DNA extration for human microbe samples.
200μl samples were mixed with 500 μl TE buffer and 20 μl of 10 mg/ml lysozyme solution.
Incubate at 37 °C for 90min. Invert the tube several times to mix every 15 minutes. Centrifuge at 13,000 rcf/min for 10 min at 4 °C and discard the supernatant.
Add 300 μl of 20 mg/ml proteinase K and 5μl of 10% SDS. Vortex to resuspend cells.
Incubate at 55 °C in a shaking water bath for 120 min to effect complete lysis. Invert the tube several times to mix every 15 minutes.
Centrifuge at 13,000 rcf/min for 10 min at 4 °C, then transfer the supernatant to a nuclease-free 2 ml microfuge tube.
Add 700 μl phenol-chloroform-isoamylalcohol (25:24:1) and vortex to mix well, centrifuge at 13,000 rcf/min for 10 min at 4 °C, then transfer the supernatant to a nuclease-free 2 ml microfuge tube.
Add 700 μl chloroform-isoamylalcohol (24:1) and vortex to mix well. Centrifuge at 13,000 rcf/min for 10 min at 4 °C, then transfer the supernatant to a nuclease-free 1.5 ml microfuge tube.
Add 1/10 volume of 3M sodium acetate, 2 μl of 5 mg/ml glycogen and 800 μl cold isopropanol. Incubate at -20 °C for the night.
Centrifuge at 13,000 rcf/min for 10 min at 4 °C, then discard the supernatant.
Washed with 800 μl of 70% ethanol. Centrifuge at 13,000 rcf/min for 10 min at 4 °C, then discard the supernatant.
Wash the precipitate with a second 800 μl of 70% ethanol and centrifuge as above. discard the supernatant.
Let sit for 15 min to dry the precipitate. Add 1×TE buffer to resuspend the precipitate.
Add 1μl RNase A to samples and invert tube several times to mix. Incubate at 37 °C for 30 minutes.