### Introduction This extraction method is recommended for hard to lyse organisms, such as *Mycobacterium tuberculosis*, or for bacteria that was not successfully extracted using the [Bacteria gDNA extraction for Nanopore-only Microbial Isolate Sequencing Solution (NO-MISS)](https://community.nanoporetech.com/extraction_methods/bact-gdna-no-miss) extraction method. Using this method you can expect a yield of ~15-40 ng/µl per sample with a DNA Integrity Number (DIN) of 8. Following this extraction method, you can perform a 24-plex library preparation using the [Nanopore-only Microbial Isolate Sequencing Solution (NO-MISS) - Rapid Barcoding Kit V14 (SQK-RBK114.24 or SQK-RBK114.96)](https://community.nanoporetech.com/docs/prepare/library_prep_protocols/no-miss-isolate-sequencing-rapid-barcoding-v14) protocol. This manual extraction method is a column-based extraction using the NEB Monarch Genomic DNA Purification Kit. ### Equipment and consumables __Materials__ - 5 – 10 mg cells from solid or liquid media __Consumables__ - Lysozyme human (Sigma, L1667) - Phosphate Buffered Saline (PBS), pH 7.4 (Thermo Fisher, 10010023) - TE buffer (Sigma, 8890-100ML) - Screw Cap for 2 mL Lysing Matrix tubes (MP Biomedicals, 115067005) - Glass beads, 4 mm (MP Biomedicals, 116914801) - Proteinase K (NEB, P8107AAVIAL) - Monarch® RNase A (NEB, T3018-1) - Empty FastPrep® 2mL Lysing Matrix tubes (MP Biomedicals, 115076200) - CTAB buffer (Promega, MC1411) - 5 M sodium chloride solution (Sigma, S6546) - Monarch® gDNA Elution Buffer (NEB, T3016-1) - Monarch® gDNA Binding Buffer (NEB, T3014-1) - Monarch® Collection Tubes II (NEB, T2018-1) - Monarch® gDNA Purification Columns (NEB, T3017-1) - Monarch® gDNA Wash Buffer (NEB, T3015-1) - Qubit 1x dsDNA HS Assay Kit (ThermoFisher, Q33230) - Nuclease-free water (e.g. ThermoFisher, AM9937) - Qubit™ Assay Tubes (Invitrogen, Q32856) - 1.5 ml Eppendorf DNA LoBind tubes __Equipment__ - P1000 pipette and tips - P100 pipette and tips - P200 pipette and tips - Thermal cycler - Eppendorf 5424 centrifuge (or equivalent) - Thermomixer - Vortex mixer - Qubit fluorometer (or equivalent) ### Method 1\. Prepare the lysozyme by reconstituting in TE buffer to 10 mg/ml. __TIP:__ We recommend preparing enzymes freshly on the day as enzymatic activity reduces over time once reconstituted. 2\. Prepare your input depending on your sample type: | __For *Mycobacterium tuberculosis*__ | | --- | | Harvest 5-10 mg of cells and transfer to a 1.5 ml Eppendorf DNA LoBind tube.
We recommend to avoid overgrowing the cultures. | | __· For *Mycobacterium tuberculosis* solid media:__ ~2-4 weeks of growth should provide sufficient biomass.

__· For *Mycobacterium tuberculosis* liquid media:__ ~ 2 weeks of growth should provide sufficient biomass.
Centrifuge ~500 µl at 12,000 x g for 1 minute and pipette off as much liquid as possible before weighing the pellet.
| |__IMPORTANT:__ For *Mycobacterium tuberculosis*, we strongly advise harvesting the recommended amount of input material to yield the correct amount of gDNA for the library preparation step.

· Less than 5 mg will not yield enough gDNA for library preparation (less than 10 ng/µl), negatively impacting sequencing output.
· More than 10 mg may result in carryover of sequencing inhibitors onto the flow cell, also negatively impacting sequencing output.| | __For other bacterial organisms__ | | --- | | __· For bacteria in solid media:__ In a fresh 1.5 ml Eppendorf DNA LoBind tube collect 1/8 of a 10 µl loop of colonies from a plate.

__· For bacteria in liquid media:__ In a fresh 1.5 ml Eppendorf DNA LoBind tube collect 200 µl (~1 x 10^8 – 10^9 cfu/ml) of cell culture.
Centrifuge at 12,000 x g for 1 minute and pipette off as much supernatant as possible.
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3\. Resuspend the pellet in 500 µl PBS and pipette mix using a P1000 pipette.
If the pipette tip becomes clogged, gently press the pipette tip down on the bottom of the tube to break up clumps while pipetting up and down. __IMPORTANT:__ We do not recommend increasing the temperature of the next incubation as this may negatively impact DNA quality and sequencing data. 4\. Incubate the sample at 80°C for 20 minutes.
This heated incubation is important for efficient cell lysis. 5\. Centrifuge at 12,000 x g for 1 minute. 6\. Remove the supernatant and resuspend the pellet in 500 µl PBS by pipette mixing using a P1000 pipette.
Washing in PBS removes any potential inhibitors from the cell suspension, as well as free DNA that may be degraded. 7\. Remove the supernatant and resuspend in 100 µl of TE buffer (10 mM Tris, pH 8 and 1 mM EDTA). 8\. Transfer 100 µl of your sample to an empty 2 ml FastPrep lysing matrix tube and add two 4 mm glass beads. 9\. Vortex the sample for 30 seconds.
Vortexing with beads helps homogenise the sample to improve efficiency of later steps. Using 4 mm beads avoids overshearing of the DNA. 10\. Add 10 µl lysozyme and mix by pulse vortexing.
Light agitation prevents aggregation and precipitation in the reaction and allows the enzyme to access the cell walls. __IMPORTANT:__ Do not extend the next incubation as this may lead to degradation and loss of DNA. 11\. Incubate at 37°C for 15 minutes at 800 RPM in a thermomixer.
__Note:__ Constant shaking is essential to facilitate cell lysis and occasional aggitation helps prevent the cells from clumping.
If a thermomixer is not available, briefly vortex at regular intervals of 2 minutes. 12\. Transfer 110 µl of your sample into a fresh 1.5 ml Eppendorf DNA LoBind tube.
Only take liquid forwards when transferring the sample. Any clumps of debris stuck to the side of the tube can be left. 13\. Combine the following reagents with your sample in the 1.5 ml Eppendorf DNA LoBind tube, and mix by vortexing. | Reagent | Volume | | --- | --- | | Proteinase K | 10 µl | | RNase A | 3 µl | | __Total (including your sample)__ | __123 µl__ | Vortex mixing your sample before adding the CTAB buffer allows the enzymes to fully mix with the bacterial cells as the mixture can become viscous. 14\. Add 200 µl of CTAB buffer and mix by vortexing.
Extractions performed using the CTAB buffer improves sequencing health and output compared to using NEB Lysis Buffer from the Monarch Genomic Purification Kit. 15\. Incubate at 56°C for 30 minutes at 1000 RPM in a thermomixer. 16\. Add 100 µl 5 M sodium chloride and centrifuge at 17,000 x g for 5 minutes.
The high salt step reduces yield but is essential to remove any inhibitors of the library preparation. 17\. Retain 200 µl of the lysate, taking care to avoid the pellet.
It is cruicial to avoid the pellet when pipetting off the lysate. __INFO:__ The NEB Monarch Genomic DNA Purification Kit is used for gDNA extraction. The remainder of this method is written following the Genomic DNA Binding and Elution protocol with our recommended alterations. 18\. Preheat the Monarch® gDNA Elution Buffer to 60°C for later use. __IMPORTANT:__ __The ratio of binding buffer to lysate (2:1) is important for the column binding efficiency.__
We recommend taking through 200 µl of lysate for use with 400 µl of binding buffer per sample.
To run more samples from the same isolate, the volumes can be increase in relation to the 2:1 ratio of buffer to lysate. 19\. Combine the following reagents in a clean 1.5 ml Eppendorf DNA LoBind tube: | Reagent | Volume | | --- | --- | | Lysate from the previous step | 200 µl | | Monarch® gDNA Binding Buffer | 400 µl | | __Total__ | __600 µl__ |
20\. Ensure the reaction is thoroughly mixed by pipetting until the liquid is homogenous.
Thorough mixing is essential to ensure the DNA binds properly to the column and to homogenise the CTAB and Monarch® gDNA Binding Buffer which solidify on initial contact. 21\. Transfer the reaction to a Monarch® gDNA purification column pre-inserted into a Monarch® collection tube, without touching the upper column area. 22\. Close the tube and centrifuge for 3 minutes at 1,000 x g to bind the gDNA and then for 1 minute at maximum speed (>12,000 x g) to clear the membrane.
Do not empty or remove the collection tube from the centrifuge when changing settings. 23\. Remove the gDNA purification column from the collection tube and discard the tube with the flow-through. 24\. Transfer the gDNA purification column to a new collection tube and add 500 µl Monarch® gDNA Wash Buffer. __IMPORTANT:__ Do NOT vortex. 25\. Close the collection tube and invert multiple times until the wash buffer reaches the cap. 26\. Immediately centrifuge the collection tube for 1 minute at maximum speed (>12,000 x g). Remove the gDNA purification column from the collection tube to discard the flow-through. 27\. Re-insert the gDNA purification column into the collection tube and add 500 µl gDNA wash buffer and close the tube. 28\. Immediately centrifuge the collection tube for 1 minute at maximum speed (>12,000 x g). Remove the gDNA purification column from the collection tube to discard the flow-through. 29\. After centifugation, ensure there is no liquid in the gDNA purification column above or below the silica membrane. If there is liquid, repeat the centrifugation until all liquid is removed. __IMPORTANT:__ Preheating the Monarch® gDNA Elution Buffer to 60°C is essential for maximum recovery of larger gDNA fragments. 30\. Place the gDNA purification column in a DNase-free 1.5 ml microfuge tube and add 100 µl of preheated Monarch® gDNA Elution Buffer. Close the tube and incubate at room temperature for 1 minute. 31\. Centrifuge for 1 minute at maximum speed (>12,000 x g) to elute the gDNA into the 1.5 ml microfuge tube.
The gDNA purification column can be discarded.
__CHECKPOINT:__ Quantify 1 µl of eluted sample using a Qubit fluorometer.
__Expected yield is ~15-40 ng/µl per sample with a DNA Integrity Number (DIN) of 8.__
Further expected results: - Nanodrop: 15-40 ng/µl - 260/280 of 1.7-1.9 - 260/230 of 2.0+
__END OF STEP:__ Take forward 200 ng per sample into the library preparation step of the [Nanopore-only Microbial Isolate Sequencing Solution (NO-MISS) - Rapid Barcoding Kit V14 (SQK-RBK114.24 or SQK-RBK114.96)](https://community.nanoporetech.com/docs/prepare/library_prep_protocols/no-miss-isolate-sequencing-rapid-barcoding-v14) end-to-end protocol.
### Results Data for extraction method yields coming soon. #### Change log | Version | Change | | --- | --- | | v1 | Initial publication |

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Hard to lyse organisms gDNA extraction for Nanopore-only Microbial Isolate Sequencing Solution (NO-MISS)

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