Sep 07, 2023

Public workspaceHMW gDNA extraction from prokaryotic cultures and cryo preservation stocks

DOI
dx.doi.org/10.17504/protocols.io.3byl4q7nrvo5/v1
  • 1University of Regensburg
Richard RKS Stöckl
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DOI: dx.doi.org/10.17504/protocols.io.3byl4q7nrvo5/v1
Protocol Citation: Richard RKS Stöckl 2023. HMW gDNA extraction from prokaryotic cultures and cryo preservation stocks. protocols.io https://dx.doi.org/10.17504/protocols.io.3byl4q7nrvo5/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 regularly use this protocol to extract gDNA suitable for Nanopore sequencing from archaea and bacteria
Created: July 06, 2023
Last Modified: September 07, 2023
Protocol Integer ID: 84589
Keywords: long-read sequencing, nanopore sequencing, gDNA, DNA extraction, HMW gDNA, HMW DNA, prokaryotic cells, archaea, bacteria
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Abstract
This protocol can be used to extract High Molecular Weight gDNA from bacterial and archaeal cultures and cryo preservations thereof.
The resulting gDNA is usually suitable for long-read sequencing and is regularly used for genome assembly following Nanopore Sequencing.
It has been tested with a variety of mostly archaeal but also bacterial strains, including, but not limited to, Thermococcales, Thermotogales, E. coli, and Desulfurococcales.
Protocol materials
ReagentSDSRothCatalog #CN30.3
Step 4
ReagentRoti-Aqua-P/C/ICarl RothCatalog #X985.2
Step 12
ReagentNuclease-free Water - 25 mlNew England BiolabsCatalog #B1500S
Step 19
ReagentBuffer EBQiagenCatalog #19086
Step 19
ReagentLysozyme from chicken egg whiteMerck MilliporeSigma (Sigma-Aldrich)Catalog #L6876
Step 2
Reagent2-PropanolMerck MilliporeSigma (Sigma-Aldrich)Catalog #190764
Step 14
ReagentCTAB (Cetyltrimethylammonium-bromide)Serva, GermanyCatalog #16530
Step 9
ReagentProteinase K, Molecular Biology Grade - 2 mlNew England BiolabsCatalog #P8107S
Step 4
ReagentSodium acetate trihydrateCarl RothCatalog #3856.1
Step 14
ReagentEthanolP212121Catalog #BE-BDH1156
Step 17
ReagentTE Buffer
In 2 steps
ReagentSodium chlorideP212121
In 2 steps
ReagentRoti-C/ICarl RothCatalog #X984.2
In 2 steps
Reagent1.5 mL LoBind tubes EppendorfCatalog #022431021
Step 13
ReagentMonarch RNase A – 1 ml (2x0.5ml)New England BiolabsCatalog #T3018L
Step 4
Safety warnings
Take appropriate precautions when handling phenol containing solutions!
Prepare cultures or cryopreservation capillaries
Prepare cultures or cryopreservation capillaries
Transfer the bacteria-/archaea-suspension (~Amount50 µL ) from one cryo preservation capillary to a 1.5 mL reaction tube or pellet a well-grown culture by centrifugation Centrifigation15000 rpm, 00:15:00 , discarding the supernatant, and resuspending the cell pellet in ~Amount50 µL media

15m
Open up the cells
Open up the cells
Add Amount490 µL ReagentTE BufferContributed by users + Amount20 µL freshly prepared ReagentLysozyme from chicken egg whiteMerck MilliporeSigma (Sigma-Aldrich)Catalog #L6876 solution (Concentration10 mg/mL , in ReagentTE BufferContributed by users ) and vortex briefly

incubate Duration00:30:00 at Temperature37 °C

30m
Incubation
add Amount5 µL ReagentProteinase K, Molecular Biology Grade - 2 mlNew England BiolabsCatalog #P8107S (Concentration20 mg/mL ) + Amount10 µL ReagentMonarch RNase A – 1 ml (2x0.5ml)New England BiolabsCatalog #T3018L (Concentration10 mg/mL ) and Amount15 µL of Concentration20 Mass / % volume ReagentSDSRothCatalog #CN30.3 , vortex briefly

Toxic
incubate Duration01:00:00 at Temperature56 °C

1h
Incubation
freeze at Temperature-80 °C for Duration00:30:00 and thaw at Temperature60 °C for Duration00:10:00

40m
Pause
Temperature
repeat Go togo to step #6 two additional times (total of three cycles)

add Amount100 µL of Concentration5 Molarity (M) ReagentSodium chlorideP212121 and mix well
Note
(this step is crucial as a CTAB–nucleic acid precipitate will form if salt concentration drops below about 0.5 M at room temperature)

Critical
add Amount80 µL of Concentration10 Mass / % volume ReagentCTAB (Cetyltrimethylammonium-bromide)Serva, GermanyCatalog #16530 in Concentration700 millimolar (mM) ReagentSodium chlorideP212121

incubate at Temperature65 °C for Duration00:30:00

30m
Incubation
Remove non-DNA components
Remove non-DNA components
34m
34m
add one volume (should be roughly Amount750 µL ) chloroform:isoamyl alcohol (24:1; ReagentRoti-C/ICarl RothCatalog #X984.2 ), shake vigorously, centrifuge full speed (> Centrifigation12.000 rcf, Room temperature, 00:02:00 ), transfer top (aqueous) phase to new tube

2m
Toxic
mix aqueous phase with equal volume phenol:chloroform:isoamyl alcohol (25:24:1; ReagentRoti-Aqua-P/C/ICarl RothCatalog #X985.2 ), centrifuge full speed (>Centrifigation12.000 rcf, Room temperature, 00:02:00 ) as before, and transfer top (aqueous) phase to new tube

2m
Toxic
mix aqueous phase with equal volume chloroform:isoamyl alcohol (24:1; ReagentRoti-C/ICarl RothCatalog #X984.2 ), centrifuge as before (>Centrifigation12.000 rcf, Room temperature, 00:02:00 ), and transfer top (aqueous) phase to new Reagent1.5 mL LoBind tubes EppendorfCatalog #022431021

2m
Toxic
Pellet and wash gDNA
Pellet and wash gDNA
34m
34m
add 0.6 volumes (roughly Amount360 µL ) of cold 100% Reagent2-PropanolMerck MilliporeSigma (Sigma-Aldrich)Catalog #190764 + 0.06 volumes (roughly Amount36 µL ) of Concentration3 Molarity (M) ReagentSodium acetate trihydrateCarl RothCatalog #3856.1 Ph5.2

incubate at Temperature-20 °C overnight

Overnight
centrifuge Centrifigation16.000 rcf, Room temperature, 00:20:00 , discard supernatant, air dry briefly

20m
add Amount0.6 volumes cold (Temperature-20 °C ) Concentration70 % (v/v) ReagentEthanolP212121Catalog #BE-BDH1156 , centrifuge at Centrifigation16.000 rcf, Room temperature, 00:10:00 , discard supernatant, air dry briefly

10m
Optional: repeat Go togo to step #17 once

resuspend in Amount50 µL ReagentNuclease-free Water - 25 mlNew England BiolabsCatalog #B1500S or ReagentBuffer EBQiagenCatalog #19086 or similar (ideally let this rest at Temperature4 °C for several hours)

Protocol references
This Protocol is partially based on methods from the following literature:
Murray, M. G. & Thompson, W. F. 1980. Rapid isolation of high molecular weight plant DNA. Nucleic Acids Research 8, 4321–4326. Zhou, J., Bruns, M. A., & Tiedje, J. M. 1996. DNA recovery from soils of diverse composition. Appl and env microbiology 62(2):316-322. Chen YY, Clancy KA, Burne RA. 1996. Streptococcus salivarius urease: genetic and biochemical characterization and expression in a dental plaque streptococcus. Infect Immun 64:585–592. Wilson, K. 2001. Preparation of Genomic DNA from Bacteria. Current Protocols in Molecular Biology 56, 2.4.1-2.4.5. Moissl-Eichinger, C. 2011. Archaea in artificial environments: their presence in global spacecraft clean rooms and impact on planetary protection. The ISME journal 5(2):209-219. Baker, J. L. 2022. Using Nanopore Sequencing to Obtain Complete Bacterial Genomes from Saliva Samples. Msystems e00491-22.