TITLE: High-molecular-weight DNA extraction from cheese rind microbial communities
AUTHORS: Emily C.P. Weiss

[DESCRIPTION]
This protocol describes extracting high-molecular-weight DNA from cheese rind microbial community samples. This DNA is suitable for Oxford Nanopore long-read sequencing.

[STEPS]
SECTION: Prepare TLB
3. 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   

SECTION: Prepare TLB
5. Cool to room temperature and then add RNase A to a final concentration of 20 μg/mL.  

SECTION: Prepare TLB
4. Heat the prepared buffer to 50 °C and vortex periodically until the lysozyme is fully dissolved.

 

SECTION: Prepare TLB
6. Filter-sterilize the TLB into a sterile container using a 0.22 μm filter.


SECTION: Equipment setup
1. Cool a centrifuge to 4 °C. It should be able to reach 17,000 × g.

 

SECTION: Equipment setup
2. Set a water bath or standing incubator to 37 °C and another to 50 °C.
SECTION: Lysis and digestion
7. Add 4 mL of prepared TLB to a 15 mL conical tube for each sample being extracted.

 

SECTION: Lysis and digestion
9. 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.
SECTION: Lysis and digestion
8. Add a small amount of liquid nitrogen into the mortar and place the pestle into the mortar to pre-chill.
SECTION: Lysis and digestion
10. Keep adding liquid nitrogen followed by grinding until the sample is a fine powder.
SECTION: Lysis and digestion
11. Transfer the rind powder into the 15 mL conical tube containing 4 mL of TLB and vortex until well mixed.
SECTION: Lysis and digestion
13. Incubate the tube(s) at 37 °C for 1 h with swirling to mix about every 15 min.
SECTION: Lysis and digestion
12. Repeat until all samples are in TLB.

 

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

SECTION: Extraction
15. Add an equal volume (4 mL) of phenol:chloroform:isoamyl alcohol and vortex for five seconds.  
SECTION: Extraction
16. Rotate on a culture wheel or hula mixer for 10 min.
SECTION: Extraction
17. Centrifuge for 10 min at 8,000 rpm at 4 °C.
SECTION: Extraction
18. Transfer the upper aqueous layer to a new 15 mL conical tube without disturbing the interphase.
SECTION: Extraction
19. Add an equal volume of phenol:chloroform:isoamyl alcohol to this aqueous layer.
SECTION: Extraction
20. Rotate on a culture wheel or hula mixer for 10 min.
SECTION: Extraction
21. Centrifuge for 10 min at 8,000 rpm at 4 °C.
SECTION: Extraction
22. Transfer the upper aqueous layer to a new 15 mL conical tube without disturbing the interphase.
SECTION: Extraction
23. Repeat steps 19–22 as necessary until the aqueous layer is no longer cloudy. 
SECTION: Extraction
24. After obtaining a clear aqueous layer, add an equal volume of chloroform to this aqueous layer. 
SECTION: Extraction
25. Rotate on a culture wheel or hula mixer for 5 min.
SECTION: Extraction
26. Centrifuge for 5 min at 8,000 rpm at 4 °C.
SECTION: Extraction
27. Transfer the upper aqueous layer to a new 15 mL conical tube without disturbing the interphase.
SECTION: Precipitation
28. Add an equal volume of ice-cold isopropanol and 0.1 volume of 3 M sodium acetate (sterile) to the aqueous layer.
SECTION: Precipitation
29. Place at −80 °C for 10 min.
SECTION: Precipitation
30. Centrifuge samples at 17,000 × g for 3 min at 4 °C.
SECTION: Precipitation
31. Remove supernatant, taking care not to remove the pelleted DNA.
SECTION: Precipitation
32. Wash pellet with 1 mL ice-cold, 70% ethanol.
SECTION: Precipitation
33. Centrifuge samples at 17,000 × g for 3 min at 4 °C.
SECTION: Precipitation
34. Remove ethanol, taking care not to remove the pelleted DNA.
SECTION: Precipitation
35. Do a quick spin to pellet any remaining ethanol and remove residual ethanol by pipetting.
SECTION: Precipitation
36. Leave the tube open to dry for about 15 min or until all ethanol has evaporated.
SECTION: Precipitation
37. Add 250 μL of molecular biology grade water to the DNA pellet and leave at room temperature overnight. 

 

SECTION: Precipitation
38. 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.