High-molecular-weight DNA extraction from cheese rind microbial communities

Cool a centrifuge to 4 °C. It should be able to reach 17,000 × g.

Set a water bath or standing incubator to 37 °C and another to 50 °C.

Prepare a minimum of 4 mL of Tris Lysis Buffer (TLB) per extraction sample.

Final concentrations:

• 10 mM Tris-Cl, pH 8
• 100 mM EDTA, pH 8
• 1% SDS
• 10 mg/mL lysozyme

Heat the prepared buffer to 50 °C and vortex periodically until the lysozyme is fully dissolved.

Cool to room temperature and then add RNase A to a final concentration of 20 μg/mL.

Filter-sterilize the TLB into a sterile container using a 0.22 μm filter.

Add 4 mL of prepared TLB to a 15 mL conical tube for each sample being extracted.

Add a small amount of liquid nitrogen into the mortar and place the pestle into the mortar to pre-chill.

Add 250 mg of the harvested cheese rind biofilm into the liquid nitrogen in the mortar and slowly use the pestle to grind the rind. Be careful to not splash the liquid nitrogen out of the mortar when first breaking the sample apart.

Keep adding liquid nitrogen followed by grinding until the sample is a fine powder.

Transfer the rind powder into the 15 mL conical tube containing 4 mL of TLB and vortex until well mixed.

Repeat until all samples are in TLB.

Incubate the tube(s) at 37 °C for 1 h with swirling to mix about every 15 min.

Add 50 μL of Proteinase K and incubate for 1 h at 50 °C with swirling every 15 min.

Add an equal volume (4 mL) of phenol:chloroform:isoamyl alcohol and vortex for five seconds.

Rotate on a culture wheel or hula mixer for 10 min.

Centrifuge for 10 min at 8,000 rpm at 4 °C.

Transfer the upper aqueous layer to a new 15 mL conical tube without disturbing the interphase.

Add an equal volume of phenol:chloroform:isoamyl alcohol to this aqueous layer.

Rotate on a culture wheel or hula mixer for 10 min.

Centrifuge for 10 min at 8,000 rpm at 4 °C.

Transfer the upper aqueous layer to a new 15 mL conical tube without disturbing the interphase.

Repeat steps 19–22 as necessary until the aqueous layer is no longer cloudy.

After obtaining a clear aqueous layer, add an equal volume of chloroform to this aqueous layer.

Rotate on a culture wheel or hula mixer for 5 min.

Centrifuge for 5 min at 8,000 rpm at 4 °C.

Transfer the upper aqueous layer to a new 15 mL conical tube without disturbing the interphase.

Add an equal volume of ice-cold isopropanol and 0.1 volume of 3 M sodium acetate (sterile) to the aqueous layer.

Place at −80 °C for 10 min.

Centrifuge samples at 17,000 × g for 3 min at 4 °C.

Remove supernatant, taking care not to remove the pelleted DNA.

Wash pellet with 1 mL ice-cold, 70% ethanol.

Centrifuge samples at 17,000 × g for 3 min at 4 °C.

Remove ethanol, taking care not to remove the pelleted DNA.

Do a quick spin to pellet any remaining ethanol and remove residual ethanol by pipetting.

Leave the tube open to dry for about 15 min or until all ethanol has evaporated.

Add 250 μL of molecular biology grade water to the DNA pellet and leave at room temperature overnight.

The following day, make sure the DNA is fully resuspended before performing any desired quality checks. If not using immediately, store DNA at −20 °C until needed.