Mar 09, 2023

Public workspaceHigh-molecular-weight DNA extraction from cheese rind microbial communities

DOI
dx.doi.org/10.17504/protocols.io.rm7vzbkj8vx1/v1
  • 1Arcadia Science
  • Arcadia Science
Arcadia Science
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DOI: dx.doi.org/10.17504/protocols.io.rm7vzbkj8vx1/v1
Protocol Citation: Emily C.P. Weiss 2023. High-molecular-weight DNA extraction from cheese rind microbial communities. protocols.io https://dx.doi.org/10.17504/protocols.io.rm7vzbkj8vx1/v1
Manuscript citation:
Borges A, Dutton R, McDaniel E, Reiter T, Weiss ECP. (2023). Paired long- and short-read metagenomics of cheese rind microbial communities at multiple time points. https://doi.org/10.57844/arcadia-0zvp-xz86
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: December 12, 2022
Last Modified: January 29, 2024
Protocol Integer ID: 73898
Keywords: HMW DNA, HMW, DNA, genome, genomic, extract, cheese, extraction, rind, microbial community, microbes, metagenomics, long-read, sequencing, genomes
Abstract
This protocol describes extracting high-molecular-weight DNA from cheese rind microbial community samples. This DNA is suitable for Oxford Nanopore long-read sequencing.
Materials
Mortar
Pestle
Liquid nitrogen
15 mL conical tubes
Centrifuge that is able to cool to 4 °C
Vortexer
50 °C and 37 °C incubators or water baths
Tris-Cl, pH 8
EDTA, pH 8
SDS
ReagentMonarch RNase A – 1 ml (2x0.5ml)New England BiolabsCatalog #T3018L
ReagentLysozyme from chicken egg whiteSigma AldrichCatalog #L6876
ReagentProteinase K, Molecular Biology Grade - 2 mlNew England BiolabsCatalog #P8107S
ReagentUltraPure™ Phenol:Chloroform:Isoamyl Alcohol (25:24:1, v/v)Thermo Fisher ScientificCatalog #15593031
Ice-cold isopropanol
3 M sodium acetate
Ice-cold 70% ethanol
Molecular biology grade water
Protocol materials
ReagentLysozyme from chicken egg whiteMerck MilliporeSigma (Sigma-Aldrich)Catalog #L6876
ReagentProteinase K, Molecular Biology Grade - 2 mlNew England BiolabsCatalog #P8107S
ReagentUltraPure™ Phenol:Chloroform:Isoamyl Alcohol (25:24:1, v/v)Thermo Fisher ScientificCatalog #15593031
ReagentMonarch RNase A – 1 ml (2x0.5ml)New England BiolabsCatalog #T3018L
ReagentLysozyme from chicken egg whiteMerck MilliporeSigma (Sigma-Aldrich)Catalog #L6876
ReagentMonarch RNase A – 1 ml (2x0.5ml)New England BiolabsCatalog #T3018L
ReagentProteinase K, Molecular Biology Grade - 2 mlNew England BiolabsCatalog #P8107S
ReagentUltraPure™ Phenol:Chloroform:Isoamyl Alcohol (25:24:1, v/v)Thermo Fisher ScientificCatalog #15593031
Equipment setup
Equipment setup
Cool a centrifuge to 4 °C. It should be able to reach 17,000 × g.

Note
This protocol describes centrifugation steps using 15 mL conical tubes. You may need to adjust volumes or use different tubes depending on the centrifuge you have available.

3m
Set a water bath or standing incubator to 37 °C and another to 50 °C.
3m
Prepare TLB
Prepare TLB
1h 16m
1h 16m
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 ReagentLysozyme from chicken egg whiteSigma AldrichCatalog #L6876

15m
Heat the prepared buffer to 50 °C and vortex periodically until the lysozyme is fully dissolved.

Note
Dissolving lysozyme at this concentration is time-consuming and requires quite a bit of vortexing. While not validated, it is possible that lower levels of lysozyme may be more ideal.

1h
Cool to room temperature and then add RNase A to a final concentration of 20 μg/mL.ReagentMonarch RNase A – 1 ml (2x0.5ml)New England BiolabsCatalog #T3018L

2m
Filter-sterilize the TLB into a sterile container using a 0.22 μm filter.


5m
Lysis and digestion
Lysis and digestion
3h 15m
3h 15m
Add 4 mL of prepared TLB to a 15 mL conical tube for each sample being extracted.

Note
This volume is for ~250 mg of cheese rind. Adjust volumes as needed depending on the amount of sample.

5m
Add a small amount of liquid nitrogen into the mortar and place the pestle into the mortar to pre-chill.
1m
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.
3m
Keep adding liquid nitrogen followed by grinding until the sample is a fine powder.
6m
Transfer the rind powder into the 15 mL conical tube containing 4 mL of TLB and vortex until well mixed.
1m
Repeat until all samples are in TLB.

Note
Time required will depend on the number of samples you are processing.

Incubate the tube(s) at 37 °C for 1 h with swirling to mix about every 15 min.
1h
Add 50 μL of Proteinase K and incubate for 1 h at 50 °C with swirling every 15 min.ReagentProteinase K, Molecular Biology Grade - 2 mlNew England BiolabsCatalog #P8107S

1h 10m
Extraction
Extraction
1h 5m
1h 5m
Add an equal volume (4 mL) of phenol:chloroform:isoamyl alcohol and vortex for five seconds.ReagentUltraPure™ Phenol:Chloroform:Isoamyl Alcohol (25:24:1, v/v)Thermo Fisher ScientificCatalog #15593031
5m
Rotate on a culture wheel or hula mixer for 10 min.
10m
Centrifuge for 10 min at 8,000 rpm at 4 °C.
10m
Transfer the upper aqueous layer to a new 15 mL conical tube without disturbing the interphase.
5m
Add an equal volume of phenol:chloroform:isoamyl alcohol to this aqueous layer.
5m
Rotate on a culture wheel or hula mixer for 10 min.
10m
Centrifuge for 10 min at 8,000 rpm at 4 °C.
10m
Transfer the upper aqueous layer to a new 15 mL conical tube without disturbing the interphase.
5m
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.
5m
Rotate on a culture wheel or hula mixer for 5 min.
5m
Centrifuge for 5 min at 8,000 rpm at 4 °C.
5m
Transfer the upper aqueous layer to a new 15 mL conical tube without disturbing the interphase.
5m
Precipitation
Precipitation
50m
50m
Add an equal volume of ice-cold isopropanol and 0.1 volume of 3 M sodium acetate (sterile) to the aqueous layer.
5m
Place at −80 °C for 10 min.
10m
Centrifuge samples at 17,000 × g for 3 min at 4 °C.
Remove supernatant, taking care not to remove the pelleted DNA.
5m
Wash pellet with 1 mL ice-cold, 70% ethanol.
2m
Centrifuge samples at 17,000 × g for 3 min at 4 °C.
3m
Remove ethanol, taking care not to remove the pelleted DNA.
2m
Do a quick spin to pellet any remaining ethanol and remove residual ethanol by pipetting.
1m
Leave the tube open to dry for about 15 min or until all ethanol has evaporated.
15m
Add 250 μL of molecular biology grade water to the DNA pellet and leave at room temperature overnight.

Note
To maximize DNA length, it is best to minimize pipetting of the DNA to avoid shearing. When pipetting, using a pipette tip with the end of the tip cut off to make it wider. This can help decrease shearing.

2m
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.

Note
Genomic DNA ScreenTape Analysis on an Agilent Tapestation can help assess quality of genomic DNA. Oxford Nanopore Community pages provide additional recommendations for DNA quality checks to perform prior to ONT sequencing.

ReagentGenomic DNA ScreenTapeAgilent TechnologiesCatalog #5067-5365 ReagentGenomic DNA ReagentsAgilent TechnologiesCatalog #5067-5366


5m