Sep 07, 2023
Version 1
HMW gDNA extraction from prokaryotic cultures and cryo preservation stocks V.1
- Richard RKS Stöckl1
- 1University of Regensburg
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, hmw gdna extraction from prokaryotic culture, hmw gdna extraction, high molecular weight gdna, following nanopore sequencing, resulting gdna, cryo preservation, cryo preservation stocks this protocol, bacterial strain, archaeal culture, prokaryotic culture, genome assembly, cryo preservation stock, genome, desulfurococcale, read sequencing, thermococcale, extraction
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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
TE Buffer
Lysozyme from chicken egg whiteMerck MilliporeSigma (Sigma-Aldrich)Catalog #L6876
SDSRothCatalog #CN30.3
Monarch RNase A – 1 ml (2x0.5ml)New England BiolabsCatalog #T3018L
Proteinase K, Molecular Biology Grade - 2 mlNew England BiolabsCatalog #P8107S
Sodium chlorideP212121
CTAB (Cetyltrimethylammonium-bromide)Serva, GermanyCatalog #16530
Roti-C/ICarl RothCatalog #X984.2
Roti-Aqua-P/C/ICarl RothCatalog #X985.2
1.5 mL LoBind tubes EppendorfCatalog #022431021
Sodium acetate trihydrateCarl RothCatalog #3856.1
2-PropanolMerck MilliporeSigma (Sigma-Aldrich)Catalog #190764
EthanolP212121Catalog #BE-BDH1156
Nuclease-free Water - 25 mlNew England BiolabsCatalog #B1500S
Buffer EBQiagenCatalog #19086
Troubleshooting
Safety warnings
Take appropriate precautions when handling phenol containing solutions!
Prepare cultures or cryopreservation capillaries
Transfer the bacteria-/archaea-suspension (~50 µL ) from one cryo preservation capillary to a 1.5 mL reaction tube or pellet a well-grown culture by centrifugation 15000 rpm, 00:15:00 , discarding the supernatant, and resuspending the cell pellet in ~50 µL media
15m
Open up the cells
Add 490 µL TE Buffer + 20 µL freshly prepared Lysozyme from chicken egg whiteMerck MilliporeSigma (Sigma-Aldrich)Catalog #L6876 solution (10 mg/mL , in TE Buffer ) and vortex briefly
incubate 00:30:00 at 37 °C
30m
add 5 µL Proteinase K, Molecular Biology Grade - 2 mlNew England BiolabsCatalog #P8107S (20 mg/mL ) + 10 µL Monarch RNase A – 1 ml (2x0.5ml)New England BiolabsCatalog #T3018L (10 mg/mL ) and 15 µL of 20 Mass / % volume SDSRothCatalog #CN30.3 , vortex briefly
incubate 01:00:00 at 56 °C
1h
freeze at -80 °C for 00:30:00 and thaw at 60 °C for 00:10:00
40m
repeat two additional times (total of three cycles)
add 100 µL of 5 Molarity (M) Sodium 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)
add 80 µL of 10 Mass / % volume CTAB (Cetyltrimethylammonium-bromide)Serva, GermanyCatalog #16530 in 700 millimolar (mM) Sodium chlorideP212121
incubate at 65 °C for 00:30:00
30m
Remove non-DNA components
34m
add one volume (should be roughly 750 µL ) chloroform:isoamyl alcohol (24:1; Roti-C/ICarl RothCatalog #X984.2 ), shake vigorously, centrifuge full speed (> 12.000 rcf, Room temperature, 00:02:00 ), transfer top (aqueous) phase to new tube
2m
mix aqueous phase with equal volume phenol:chloroform:isoamyl alcohol (25:24:1; Roti-Aqua-P/C/ICarl RothCatalog #X985.2 ), centrifuge full speed (>12.000 rcf, Room temperature, 00:02:00 ) as before, and transfer top (aqueous) phase to new tube
2m
mix aqueous phase with equal volume chloroform:isoamyl alcohol (24:1; Roti-C/ICarl RothCatalog #X984.2 ), centrifuge as before (>12.000 rcf, Room temperature, 00:02:00 ), and transfer top (aqueous) phase to new 1.5 mL LoBind tubes EppendorfCatalog #022431021
2m
Pellet and wash gDNA
34m
add 0.6 volumes (roughly 360 µL ) of cold 100% 2-PropanolMerck MilliporeSigma (Sigma-Aldrich)Catalog #190764 + 0.06 volumes (roughly 36 µL ) of 3 Molarity (M) Sodium acetate trihydrateCarl RothCatalog #3856.1 5.2
incubate at -20 °C overnight
centrifuge 16.000 rcf, Room temperature, 00:20:00 , discard supernatant, air dry briefly
20m
add 0.6 volumes cold (-20 °C ) 70 % (v/v) EthanolP212121Catalog #BE-BDH1156 , centrifuge at 16.000 rcf, Room temperature, 00:10:00 , discard supernatant, air dry briefly
10m
Optional: repeat once
resuspend in 50 µL Nuclease-free Water - 25 mlNew England BiolabsCatalog #B1500S or Buffer EBQiagenCatalog #19086 or similar (ideally let this rest at 4 °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.