DNA extraction: Shortened Godon protocol with optional enzymatic lysis

Metadata

Title	Category	Adapated and used by	last accessed	Link to protocol	Primary origin for protocol
DNA extraction: Shortened Godon protocol with optional enzymatic lysis	DNA extraction	University Hospital of RWTH Aachen, Inst. Med. Microbiology, Clavel lab
Collaborative Research Center 1382, Project Q02 “Integrative Microbiota Analysis”, Dr. Nicole Treichel	2024-09-12	(Treichel, 2022)	(“Molecular Microbial Diversity of an Anaerobic Digestor as Determined by Small-Subunit RDNA
Sequence Analysis,” 1997)</a>

Consider accessing the protocol for a donwloadable PDF version.

Protocol

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Prepare:

• TE buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA)
• Bead-beater-tubes (with 500 mg+/- 10 mg Zirconia/Silicia beads, autoclaved)
• 4M Guanidinethiocyanate in 0,1M Tris pH 7,5 (consider appropriate disposal)
• 5 % N-laurolylsarcosine in PBS (Dulbecco’s phosphate Buffered Saline) (consider appropriate disposal)

Sample preparation:

Consider first if you want to use the enzymatic lysis step!

• For extraction from bacterial culture: Take 2 ml of a well grown culture, centrifuge it at 12.000 x g for 10 min, and discard the supernatant
• For faecal samples stored premixed 1:6 with DNA Stabilizer (recommended for protocol used without enzymatic step): let thaw at room temperature.
• For faecal samples stored without DNA Stabilizer, add DNA stabilizer (1:6) to frozen sample, mix and thaw at room temperature
• If protocol is used with enzymatic step do not use DNA Stabilizer

DNA Isolation:

For enzymatic extraction Prepare Lysozyme-Solution (TE Buffer with 15 µg/µl Lysozyme): mass Lysozyme = (#Samples(+1)) * 7.5 mg Volume TE buffer = (#Samples(+1)) * 0.5 ml Weigh lysozyme into TE buffer (leave lysozyme stock on ice!)

1. Resuspend bacterial pellet or faecal sample in 500 µl lysozyme-solution
2. Incubate for 30 min at 37 °C
3. Add 50 µl 10 % SDS
4. Add 15 µl ProteinaseK (20 mg/ml)
5. Incubate for 1-2 h at 50 °C (liquid should be totally clear) Continue with step 2 below

Without enzymatic extraction For extraction without the enzymatic step, resuspend the bacterial pellet in 600 µl DNA Stool Stabilizer or use 600µL of faecal sample in DNA stabilizer

1. Transfer sample into bead-beater-tubes (with 500 mg+/- 10 mg Zirconia/Silicia beads, previously autoclaved)
2. Add 250 µl 4M Guanidinethiocyanate
3. Add 500 µl 5 % N-laurolylsarcosine  vortex
4. Incubate at 70 °C while shaking (700 rpm) for 60 min; Set centrifuge to 4 °C
5. Bead-beat 3 x with the following program; re-fill the cooling adapter of the bead-beater with dry ice between each round: CY; 40 s; 6,6 m/s, cooled with dry ice
6. Add 15 mg PVPP [Poly(vinylpolypyrrolidone)]
   Vortex, then centrifuge at 15.000 x g, 4 °C, 3 min
7. Transfer clear supernatant into a new 2 ml tube Centrifuge at 15.000 x g, 4 °C, 3 min
8. Take 500 µl clear supernatant into a new 2 ml tube
9. Add 5 µl RNase (10 mg/ml), incubate at 37 °C while shaking (700 rpm) for 20-30 min (heat incubator afterwards to 70 °C, heat up DE)
10. Proceed with the NucleoSpin® gDNA Clean-up follow protocol

Material

• ProteinaseK; Carl Roth (7528.1)
• NucleoSpin® gDNA Clean-up; Macherey-Nagel (740230.250)
• RNase; Sigma-Aldrich (R6513-50mg)
• Guanidinethiocyanate; Sigma-Aldrich (G9277-100G)
• Zirconia/Silicia beads; Biozym (55D1132-01TP)
• N-laurolylsarcosine; Sigma-Aldrich (61743-25G)
• TE buffer; Sigma-Aldrich (T9285-100ML)
• Poly(vinylpolypyrrolidone); Sigma-Aldrich (P5288-100G)
• 1.5 mL and 2 mL Eppendorf tubes

References

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1. Treichel, N. (2022). DNA extraction: Shortened Godon protocol with optional enzymatic lysis. CRC1382. https://www.crc1382.org/wp-content/uploads/2022/11/221019_DNA-extraction.pdf
2. Molecular microbial diversity of an anaerobic digestor as determined by small-subunit rDNA sequence analysis. (1997). Appl Environ Microbiol., 63. https://doi.org/10.1128/aem.63.7.2802-2813.1997