Consider accessing the protocol for a donwloadable PDF version.
Protocol
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!)
- Resuspend bacterial pellet or faecal sample in 500 µl lysozyme-solution
- Incubate for 30 min at 37 °C
- Add 50 µl 10 % SDS
- Add 15 µl ProteinaseK (20 mg/ml)
- 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
- Transfer sample into bead-beater-tubes (with 500 mg+/- 10 mg Zirconia/Silicia beads, previously autoclaved)
- Add 250 µl 4M Guanidinethiocyanate
- Add 500 µl 5 % N-laurolylsarcosine vortex
- Incubate at 70 °C while shaking (700 rpm) for 60 min; Set centrifuge to 4 °C
- 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
- Add 15 mg PVPP [Poly(vinylpolypyrrolidone)]
Vortex, then centrifuge at 15.000 x g, 4 °C, 3 min
- Transfer clear supernatant into a new 2 ml tube
Centrifuge at 15.000 x g, 4 °C, 3 min
- Take 500 µl clear supernatant into a new 2 ml tube
- 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)
- 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