This extraction method is a universal automated DNA extraction from bacteria and fungi/yeast. It uses vortex bead-beating followed by a proteinase K and RNase treatment (other extraction platforms are available that may be used and optimised).

Using this method you can expect a yield of >200 ng (200-500 ng) per sample with a DNA Integrity Number (DIN) of 7-9 and expected fragment lengths of >15 kb from the extraction. Please note, yield will vary depending on bacterial species but >20 ng/µl is sufficient for use with the Nanopore-only Microbial Isolate Sequencing Solution (NO-MISS) from cell cultures using SQK-RBK114 (.24 or .96) end-to-end protocol.

Note: If your desired yield is not achieved or you are looking to maximise the contiguity of your assemblies by generating longer read lengths, try one of our sample-tailored extraction methods. Further details can be found for each method via the links below:

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

Materials

• Liquid overnight culture (~10^8 – 10^9 cfu/ml) or half a 10 µl loop of colonies from a plate

Consumables

• RNase A (QIAGEN, 19101)
• Proteinase K (QIAGEN, 19131)
• BashingBead Buffer (Zymo, D6001-3-40)
• PowerBead Pro tube (Qiagen, 19301)
• Phosphate Buffered Saline (PBS), pH 7.4 (Thermo Fisher, 10010023)
• MagMAX™ DNA Multi-Sample Ultra 2.0 Kit (ThermoFisher, A36570)
• Nuclease-free water (e.g. ThermoFisher, AM9937)
• 10 µl inoculating loop
• 1.5 ml Eppendorf DNA LoBind tubes
• 96-well PCR plate, semi-skirted (e.g. Starlab, I1402-9800)
• PCR plate seals
• Qubit™ Assay Tubes (Invitrogen, Q32856)
• Qubit 1x dsDNA HS Assay Kit (ThermoFisher, Q33230)

Equipment

• Vortex Adapter (Qiagen 13000-V1-24)
• Eppendorf 5424 centrifuge (or equivalent)
• Hula mixer (gentle rotator mixer)
• Qubit fluorometer (or equivalent)
• Thermomixer
• Magnetic rack
• Vortex mixer
• P1000 pipette and tips
• P200 pipette and tips
• P100 pipette and tips
• P20 pipette and tips
• P2 pipette and tips

1. Prepare your sample depending on your sample type by following the instructions below:

If using liquid media for your culture:
1. Centrifuge 1ml liquid overnight culture (~10^8 – 10^9 cfu/ml) at 10,000 xg for 1 minute
2. Remove the supernatant and resuspend the pellet in 1 ml Phosphate Buffered Saline (PBS) Note: Washing the sample by removing the supernatant and resuspending the pellet in clean Phosphate Buffered Saline (PBS) removes potential inhibitors from the nutrient broth and removes free DNA that may be degraded
3. Centrifuge at 10,000 x g 1 minute
4. Remove the supernatant, without disturbing the pellet
5. Resuspend the pellet in 350 µl bashing bead buffer (Zymo, D6001-3-40)
6. Add the 350 µl of resuspended sample to a PowerBead Pro tube (Qiagen, 19301)
7. Take forward the PowerBead Pro tube containing 350 µl of resuspended sample into the next step

If using solid media for your culture:
1. Add 350 µl BashingBead Buffer (Zymo, D6001-3-40) to a PowerBead Pro tube (Qiagen, 19301)
2. Using a sterile 10 µl inoculating loop, pick half a loop worth of colonies, avoiding scratching the agar
3. Place the inoculating loop with the colonies in the PowerBead Pro tube containing the BashingBead Buffer, and agitate to relieve the cells into the tube
4. Discard the used inoculating loop
5. Take forward the PowerBead Pro tube containing 350 µl of resuspended sample into the next step

2. Secure the PowerBead Tube(s) containing your 350 µl of resuspended sample from the previous step to a vortex mixer using a Vortex Adapter (e.g. Qiagen, 13000-V1-24).

3. Vortex at maximum speed for 5 minutes (~3000 RPM on most common vortex mixers).

4. Centrifuge the tube(s) containing your sample at 10,000 x g for 30 seconds.

5. Transfer the supernatant from the bead-beating tube into a clean 1.5 ml Eppendorf DNA LoBind tube.
Note: Expect ~250–300 µl of supernatant.

6. Add the following reagents with your sample in the 1.5 ml Eppendorf DNA LoBind tube, and mix by vortexing.

• For most bacterial inputs:

Reagent	Volume
Proteinase K	10 µl
RNase A	3 µl

• For fungal inputs:

Reagent	Volume
Proteinase K	30 µl
RNase A	3 µl

IMPORTANT: The full 15-minute incubation must be completed to ensure the proteinase and RNase activity occurs, regardless of turbidity.

7. Incubate at 56°C for 15 minutes in a thermomixer at 1000 RPM.
Note: Depending on the bacterial input, the solution may become fully transparent or may remain hazy.

INFO: The Maxwell RSC PureFood Pathogen Kit is used with the Maxwell RSC instrument. The remainder of the method is written following the Maxwell protocol.
The Maxwell RCS PureFood Pathogen Kit technical manual can be found here.

8. To each sample, add 400 µl Binding solution from the MagMAX™ DNA Multi-Sample Ultra 2.0 Kit, and 40 µl of the DNA binding beads.

9. Gently mix by pipetting and incubate each sample at 800rpm on a hula mixer for 5 minutes.

10. Pellet your sample(s) on a magnetic rack and carefully remove and discard the supernatant without disturbing the pellet. Remove the samples from the magnetic rack, add 1000 µl of wash solution, and resuspend the sample(s) by gently pippetting.

12. Pellet your sample(s) on a magnetic rack and carefully remove and discard the supernatant without disturbing the pellet. Remove the samples from the magnetic rack, add 1000 µl 80% ethanol, and resuspend the sample(s) by gently pippetting.

14. Pellet your sample(s) on a magnetic rack and carefully remove and discard the supernatant without disturbing the pellet. Remove the samples from the magnetic rack, add 500 µl 80% ethanol, and resuspend the sample(s) by gently pippetting.

15. Pellet your sample(s) on a magnetic rack and carefully remove and discard the supernatant without disturbing the pellet. Air dry your sample(s) for 5 minutes.

16. To each sample, add 100 µl of elution solution, resuspend the sample(s) by gently pipetting. Mix your sample(s) on a hula mixer at 800 rpm for 5 minutes.

17. Pellet your sample(s) on a magnetic rack. For each sample, carefully remove and store the clean eluate into a separate 1.5 ml Eppendorf DNA LoBind tube.

CHECKPOINT: Quantify 1 µl of eluted sample using a Qubit fluorometer.

Expected yield is >200 ng (200-500 ng) per sample with a DNA Integrity Number (DIN) of 7-9.

END OF STEP: Take forward 200 ng per sample into the library preparation step of the Nanopore-only Microbial Isolate Sequencing Solution (NO-MISS) from cell cultures using SQK-RBK114 (.24 or .96) end-to-end protocol.

Data for extraction method yields coming soon.