Introduction

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, and automated extraction using the Promega maxwell (other extraction platforms can 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 >30 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) - Rapid Barcoding Kit V14 (SQK-RBK114.24 or SQK-RBK114.96) 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)

Equipment and consumables

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)
• Maxwell® RSC PureFood Pathogen kit (Promega, AS1660)
• PowerBead Pro tube (Qiagen, 19301)
• Phosphate Buffered Saline (PBS), pH 7.4 (Thermo Fisher, 10010023)
• Nuclease-free water (e.g. ThermoFisher, AM9937)
• 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)
• Maxwell® RSC Instrument (MPBiomedicals, AS4500)
• 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

Optional equipment

• Hula mixer (gentle rotator mixer)

Method

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) countaining 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 10 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	1 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. Prepare a Maxwell RSC Cartridge for each sample in the Maxwell RSC instrument deck tray:

Steps to be performed for each sample:
1. Place the cartridge in the deck tray with the the labeled side facing away from the elution tube position.
2. Press the cartridge down to snap it into position.
3. Carefully peel back the seal to remove the plastic from the top of the cartridge. Note: Ensure that all sealing tape and any resifual adhesive is removed from the cartridge before proceeding.
Important: If you are processing fewer than 16 samples, centre the cartridges on the cartridge rack.
4. Place a Maxwell RSC Plunger into well #8 (closest well to the Elution Tube). Note: Use only the plungers provided in the Maxwell RSC PureFood Pathogen Kit.
5. Place the 0.5 ml Elution Tube in the front of the deck tray, next to well #8 of the cartridge.
6. Add 50 µl of Elution Buffer to the bottom of the empty Elution Tube.
7. Add 300 µl of the Lysis Buffer (from the Maxwell RSC PureFood Pathogen Kit) to your sample prepared for extraction (from previous step). Note: DO NOT use Lysis Buffer A.
8. Mix the sample by vortexing for 5–10 seconds.
9. Add the entire sample with the Lysis Buffer to well #1 of the cartridge.

9. Set up the Maxwell® RSC Instrument (Cat.# AS4500) to run the method for DNA purification according to instrument operating manual and start extraction.

10. After extraction has completed, remove the deck tray from the instrument.

11. Remove and close the elution tubes containing the extracted DNA.

IMPORTANT: Fungi and yeast samples processed using the universal bead-beating gDNA extraction must go through a AMPure XP bead clean-up step.
Fungi and yeast samples must undergo a 0.4X AMPure XP bead clean-up following gDNA extraction and before proceeding to library preparation to remove inhibitors present in the samples.
For more information on fungi and yeast samples please refer to our Fungi gDNA extraction for Nanopore-only Microbial Isolate Sequencing Solution (NO-MISS) extraction method.

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) - Rapid Barcoding Kit V14 (SQK-RBK114.24 or SQK-RBK114.96) end-to-end protocol.

Results

Data for extraction method yields coming soon.

Change log

Version	Change
v1	Initial publication