May 02, 2025

Public workspace High Molecular Weight DNA Extraction from Actinomycetota

DOI
dx.doi.org/10.17504/protocols.io.q26g7mqr9gwz/v1
  • 1Iowa State University
Ashley Paulsen
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DOI: dx.doi.org/10.17504/protocols.io.q26g7mqr9gwz/v1
Protocol Citation: Ashley Paulsen, Larry Halverson 2025. High Molecular Weight DNA Extraction from Actinomycetota. protocols.io https://dx.doi.org/10.17504/protocols.io.q26g7mqr9gwz/v1
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: March 05, 2025
Last Modified: May 02, 2025
Protocol Integer ID: 123879
Funders Acknowledgements:
National Science Foundation
Grant ID: DGE-1545453
National Science Foundation
Grant ID: DRL-1759152
U.S. Department of Energy (DOE) Office of Biological and Environmental Research, Biological Systems Division
Grant ID: FWP No AL-18-380-055
Abstract
Actinomycetes are difficult to extract DNA from and excluding mechanical lysis makes it especially challenging. This protocol is a modified phenol-chloroform DNA extraction for extracting high molecular weight DNA from Actinomycetes for whole genome long-read sequencing and has been verified to produce extractions with an N50 of >30 kb. The main modifications include an Actinomycetes optimized lysis buffer and a PEG-based size selective precipitation step.
Materials
Growth Medium
ReagentTryptic Soy BrothFisher ScientificCatalog #DF0370-07-5
ReagentGlycine Contributed by users

Extraction
Reagent1X PBS (Phosphate-buffered saline )
ReagentSodium chlorideP212121
ReagentSucroseP212121
ReagentTris HClP212121
ReagentEDTAContributed by users
ReagentLysozymeMerck MilliporeSigma (Sigma-Aldrich)Catalog #12671-19-1
ReagentRNase A (10 mg/mL)Thermo Fisher ScientificCatalog #EN0531
ReagentSodium Dodecyl SulfateP212121
ReagentBioUltra TE-saturated phenolMerck MilliporeSigma (Sigma-Aldrich)Catalog #77607
ReagentBioUltra Chloroform – isoamyl alcohol mixture 24:1SupelcoCatalog #25666
Reagentammonium acetateP212121
ReagentEthanolP212121Catalog #BE-BDH1156
Reagent70% Ethanol Contributed by users
Reagent10 mM Tris-HCL pH 8.0Contributed by users

Size selective precipitation
ReagentPoly(ethylene glycol) 8000 [PEG 8000]Merck MilliporeSigma (Sigma-Aldrich)Catalog #89510
Protocol materials
Reagent1X PBS (Phosphate-buffered saline )
ReagentBioUltra TE-saturated phenolMerck MilliporeSigma (Sigma-Aldrich)Catalog #77607
ReagentBioUltra Chloroform – isoamyl alcohol mixture 24:1SupelcoCatalog #25666
ReagentBioUltra TE-saturated phenolMerck MilliporeSigma (Sigma-Aldrich)Catalog #77607
Reagent1X PBS (Phosphate-buffered saline )
ReagentProteinase KLife TechnologiesCatalog #17916
Reagent10 mM Tris-HCL pH 8.0
Reagent10 mM Tris-HCL pH 8.0
Reagentammonium acetateP212121
ReagentTris HClP212121
ReagentEDTA
ReagentRNase A (10 mg/mL)Thermo Fisher ScientificCatalog #EN0531
ReagentGlycine
ReagentBioUltra Chloroform – isoamyl alcohol mixture 24:1SupelcoCatalog #25666
Reagent70% Ethanol
Reagent10 mM Tris-HCL pH 8.0
ReagentSodium chlorideP212121
ReagentTryptic Soy BrothFisher ScientificCatalog #DF0370-07-5
ReagentBioUltra TE-saturated phenolMerck MilliporeSigma (Sigma-Aldrich)Catalog #77607
ReagentLysozymeMerck MilliporeSigma (Sigma-Aldrich)Catalog #12671-19-1
Reagent1X PBS (Phosphate-buffered saline )
ReagentSucroseP212121
ReagentSodium Dodecyl SulfateP212121
Reagentammonium acetateP212121
ReagentEthanolP212121Catalog #BE-BDH1156
ReagentPoly(ethylene glycol) 8000 [PEG 8000]Merck MilliporeSigma (Sigma-Aldrich)Catalog #89510
Before start
Reagents to prepare

half strength TSB plus glycine:
  • 15 g/L Tryptic Soy Broth
  • 0.5% glycine

Sucrose Lysis Buffer:
  • 100 mM NaCl,
  • 25% (w/v) sucrose
  • 10 mM Tris-Cl pH 8
  • 25 mM EDTA pH 8
  • 1 mg/mL lysozyme
  • 20 µg/ml RNase A - added immediately before use

10% (w/v) sodium dodecyl sulfate (SDS) solution
70% Ethanol
5M ammonium acetate - prepared fresh and chilled

Elution Buffer - 10 mM Tris-HCl pH 8

PEG precipitation buffer:
  • 9% polyethylene glycol 8k (w/v)
  • 1 M NaCl
  • 10 mM Tris-HCl pH 8
Lysis
Lysis
3h 10m
3h 10m
Grow cultures shaking in half strength TSB with 0.5% glycine for 36-48 hours.


Spin down at 4500 x g for 10 minutes, remove supernatant
Centrifigation4500 x g, Room temperature, 00:10:00

10m
Wash cells by re-suspending in 500 µL of PBS, centrifuging, and removing the supernatant
Amount500 µL Reagent1X PBS (Phosphate-buffered saline )

Note
At this point the cells can be frozen and stored at Temperature-80 °C if needed


Re-suspend in Amount200 µL Reagent1X PBS (Phosphate-buffered saline )

Vortex with Amount5 mL of sucrose lysis buffer and incubate at Temperature37 °C for Duration01:00:00
Amount5 mL Sucrose Lysis Buffer

1h
Add Amount500 µL of 10% SDS (for a final concentration of 1%) and Amount50 µL ReagentProteinase KLife TechnologiesCatalog #17916 (20 mg/mL stock solution). Mix by rotating the tube end over end 5 times.


Incubate at Temperature50 °C for Duration02:00:00 , mix every 30 minutes by slowly rotating end-over-end 3 times.

2h
Extraction
Extraction
45m
45m
Pour the viscous lysate into fresh 15 mL conical centrifuge tubes
Add Amount5 mL recently opened BioUltra Phenol to each Falcon tube containing lysate.
ReagentBioUltra TE-saturated phenolMerck MilliporeSigma (Sigma-Aldrich)Catalog #77607
Note
buffer saturated phenol is preferable as the pH is around 8.0
Phenol should be used with 3 months of opening for optimum results



Place on a mixer at ~20 rpm end-over-end rotation for 10 minutes, if a fine emulsion has not formed after a minute gradually increase the rotation speed.
Note
A HulaMixer is recommended, but a hematology mixer can be used instead.

10m
Spin in a centrifuge at 6000 rpm for 10 minutes.
Centrifigation6000 rpm, Room temperature, 00:10:00

10m
Due to the sucrose, the aqueous phase will migrate below the organic. Pipette off the organic phase and discard it, removing as much as possible without removing too much of the aqueous phase. Protein may show as a white precipitate and should be removed as much as possible.
Pour the aqueous phases into new 15 ml Falcon tubes and add Amount2.5 mL of phenol and Amount2.5 mL of 24:1 chloroform-isoamyl alcohol to each.

ReagentBioUltra TE-saturated phenolMerck MilliporeSigma (Sigma-Aldrich)Catalog #77607 ReagentBioUltra Chloroform – isoamyl alcohol mixture 24:1SupelcoCatalog #25666


Place on a mixer at ~20 rpm end-over-end rotation for 10 minutes, if a fine emulsion has not formed after a minute gradually increase the rotation speed.
10m
Centrifuge at 6000 rpm for 10 minutes.
Centrifigation6000 rpm, Room temperature, 00:10:00

10m
Pipette off the organic phase and discard it, removing as much as possible without removing too much of the aqueous phase.
Centrifuge at 6000 rpm for 5 minutes and ensure complete removal of the organic phase.
Centrifigation6000 rpm, Room temperature, 00:10:00

5m
Pour aqueous phase into a new 50 mL conical centrifuge tube.
Precipitation and elution
Precipitation and elution
1d 0h 19m
1d 0h 19m
Add Amount2 mL of cold 5M ammonium acetate. Gently rock the tube back and forth 3 times to mix.
Reagentammonium acetateP212121
Note
Ammonium acetate solution should be used within a week of preparing, the fresher the better.



Add Amount15 mL ice-cold 100% ethanol and incubate tubes in Temperature4 °C DurationOvernight to maximize precipitation.

Note
Chill the ethanol before hand

Pause
Overnight
Melt the tip of a glass pipet in a bunsen burner to make a small hook
Hook out the mass of precipitated DNA and dip it into a container 70% ethanol. Try to get it in one piece, but if it breaks then you can just go back for the rest. Gently work the mass off of the hook and into a labeled microcentrifuge tube
Add Amount1 mL 70% ethanol to the tube

Spin down at 10,000 x g for 2 min then remove as much of the 70% ethanol as possible
Centrifigation10000 x g, 00:02:00

2m
Wash again with Amount1 mL 70% ethanol

Spin down at 10,000 x g for 2 min then remove as much of the 70% ethanol as possible
Centrifigation10000 x g, 00:02:00

2m
Let the remaining ethanol evaporate by leaving at room temperature for 15 minutes. Leaving the tubes on their side in a laminar flow hood speeds this up significantly.
15m
Add Amount500 µL 10 mM Tris elution buffer and incubate without mixing at Temperature4 °C for Duration24:00:00 to allow the pellet to fully re-suspend into a translucent viscous gel.
Reagent10 mM Tris-HCL pH 8.0Contributed by users

Note
This can be decreased if the pellet is small.
It’s important that freshly eluted HMW DNA is given time to relax before being handled and 4°C facilitates this relaxing.

1d
Overnight
Homogenization
Homogenization
1h
1h
1) Slowly pipette mix DNA 5-10 times with wide bore pipette (or a cut p200)
2) Incubate on Temperature37 °C heating block 30-60 minutes

1h
3) Slowly pipette mix 5-10 times with wide bore pipette
4) Perform nanodrop in triplicate; bottom, middle, top (I always use this order just for consistency) If the readings are too different then repeat the homogenization.
5) Once nanodrop readings are consistent the DNA is ready for Qubit and intended uses
Optional Size selection step
Optional Size selection step
17m
17m
Take an aliquot of DNA and add to a clean 1.5 mL micro centrifuge tube. Add an equal volume of precipitation buffer and gently flick to mix.
Incubate sample atTemperatureRoom temperature for 6-18 hours. Longer incubation times increases recovery.

Centrifuge for 15 minutes at 10,000 g
Centrifigation10000 rpm, 00:15:00

15m
Pipette off supernatant, being VERY careful not to disturb the pellet

Note
Pipette slowly, keeping tip at the liquid-air interface
Leave 10-20 µL behind

Critical
Add Amount1 mL of 70% ethanol and gently tap to mix

Centrifuge for 2 minutes at 10,000 g
Centrifigation10000 x g, 00:02:00

2m
Pipette off and discard supernatant. The pellet should be slightly more secure at this point, but care should still be taken not to disturb it.
Repeat the ethanol wash and let pellet dry for ~15 minutes.
Add desired volume of 10 mM Tris elution buffer and incubate at Temperature4 °C
Reagent10 mM Tris-HCL pH 8.0Contributed by users