Feb 10, 2025

Public workspaceBacterial Isolate Genomic DNA Isolation - ONT NO-MISS

DOI
dx.doi.org/10.17504/protocols.io.bp2l6dz4zvqe/v1
  • 1Biotechnology Teaching Program (BIT), North Carolina State University
  • BIT-Protocols
    Tech. support phone: +91 95134-135 email: ccgoller@ncsu.edu
Carlos Carlos Goller
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DOI: dx.doi.org/10.17504/protocols.io.bp2l6dz4zvqe/v1
Protocol Citation: Carlos Carlos Goller 2025. Bacterial Isolate Genomic DNA Isolation - ONT NO-MISS. protocols.io https://dx.doi.org/10.17504/protocols.io.bp2l6dz4zvqe/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 use this protocol and it's working
Created: February 09, 2025
Last Modified: February 10, 2025
Protocol Integer ID: 119848
Keywords: ONT, Nanopore, Bacterial isolates, NO-MISS
Funders Acknowledgements:
NC State Biotechnology Program
Disclaimer
This protocol was adapted from the ONT NO-MISS protocol (February 2025) and modified for teaching lab sessions.
Abstract
Overview and Goals
Your bacterial isolate has been grown on agar plates. We now need to lyse open the bacteria and isolate only the genomic DNA for sequencing. We need to lyse the bacteria with enzymes and a lysis buffer for this. We will then purify the genomic DNA. We will use a modification of the NEB Monarch Spin Column Kit extraction kit. This protocol has been recommended by Oxford Nanopore Technologies (ONT), the company that makes the sequencer we will use for sequencing our bacterial genomes.



Lysis of bacteria. Image created with BioRender.com

On the left are healthy living bacteria (An example is Bacillus). On the right, the bacteria are lysed open, which results in cell death. Consider this question as you read the protocol: How will we facilitate bacterial lysis? Images of Bacilli werecreated using BioRender.com.

Learning Objectives
After completing this lab you will gain the following lab skills:
  • Lab safety and proper personal protective equipment (PPE)
  • Proper use of a centrifuge and pipettors
  • Isolation and purification of genomic DNA
Image Attribution
Icon from The Noun Project and recolored by Carlos C. Goller
Image created using BioRender.com

Guidelines
All plastics and cultures will be treated as biohazards. Cultures will be treated with 10% (final) bleach overnight. Plastic tubes, pipettes, and spin columns used for the extraction process will be disposed of in the biohazard containers.
Materials
Supplies
We will need several supplies for this lab, including enzymes.

Materials
  • 200 µl liquid overnight culture (~1 x 108 – 1 x 109 cfu/ml) or 1/8 of a 10 µl loop of colonies from a plate


Consumables

  • Lysozyme human (Sigma, L1667)
  • Trizma‱ hydrochloride solution (Sigma, T2819)
  • 5 M sodium chloride solution (Sigma, S6546)
  • SDS at 10% v/v (Sigma, 71736)
  • Phosphate Buffered Saline (PBS), pH 7.4 (Thermo Fisher, 10010023)
  • TE buffer (Sigma, 8890-100ML)
  • Proteinase K (NEB, P8200AAVIAL) from the Monarch Spin gDNA Extraction Kit
  • Monarch‱ RNase A (NEB, T3018-1) from the Monarch Spin gDNA Extraction Kit
  • CTAB buffer (Promega, MC1411)
  • Monarch‱ gDNA Elution Buffer (NEB, T3016-1) from the Monarch Spin gDNA Extraction Kit
  • Monarch‱ gDNA Binding Buffer (NEB, T3014-1) from the Monarch Spin gDNA Extraction Kit
  • Monarch‱ Spin Collection Tubes (NEB, T2118-1) from the Monarch Spin gDNA Extraction Kit
  • Monarch‱ Spin Columns S2C and Tubes (NEB, T3017-1) from the Monarch Spin gDNA Extraction Kit
  • Monarch‱ gDNA Wash Buffer (NEB, T3015-1) from the Monarch Spin gDNA Extraction Kit
  • 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
  • 5 ml Eppendorf DNA LoBind tubes


Equipment

  • Eppendorf 5424 centrifuge (or equivalent)
  • Eppendorf Thermomixer
  • Vortex mixer
  • P1000 pipette and filter tips
  • P200 pipette and filter tips
  • P100 pipette and filter tips
  • P20 pipette and filter tips
  • P10 and P2.5 pipette and filter tips
Safety warnings
Dispose of biohazard in the appropriate biohazard bins.
Before start
Review the complete protocol before beginning. Several of the steps in this procedure are time and/or temperature-sensitive, so it’s important that you know what to expect and how to manage your time. There are also several types of tubes with specific names that need to be used for specific steps of the protocol. They are shown in the illustration below to help you keep track of these materials.

Growth of Isolates
Growth of Isolates
A colony of each of your isolates will be used to start a liquid culture in Tryptic Soy Broth (TSB).
Grow liquid cultures with shaking atTemperature30 °C for 16-18 hours.

Bacterial Lysis
Bacterial Lysis
32m
32m
For general bacterial inputs, reconstitute the lysozyme in TE buffer to 10 mg/ml. We recommend preparing enzymes freshly on the day as enzymatic activity reduces over time once reconstituted.
In a fresh 1.5 ml Eppendorf DNA LoBind tube, centrifuge Amount1 mL (~1 x 108 – 109 cfu/ml) of cell culture or 1/8 of a 10 µl loop of colonies from a plate at Centrifigation12.000 x g, 20°C, 00:01:00 .

1m
Remove the supernatant by pipetting and resuspend the pellet in Amount1 mL of PBS.

Centrifuge at Centrifigation12.000 x g, 20°C, 00:01:00 .

1m
Depending on your input, remove the supernatant and resuspend as follows:
For most bacterial inputs: Remove the supernatant and resuspend the pellet in Amount100 µL TE buffer with Amount10 µL lysozyme. This is the protocol we will follow.

For staphylococcal inputs: Remove the supernatant and resuspend the pellet in Amount100 µL Staphylococcal Lysis Buffer (SLB) withAmount10 µL achromopeptidase. Note: It is important to remove as much PBS as possible without disturbing the pellet, as the buffer has an inhibitory effect on achromopeptidase activity.

Incubate at Temperature37 °C for 10 minutes in a thermomixer at 500 RPM. Shaker500 rpm, 37°C, 00:10:00 Use Thermomixer . If a thermomixer is unavailable, the reaction can be incubated stationary and agitated by gently flicking for 10 seconds every two minutes.

Combine the following reagents with your sample in the 1.5 ml Eppendorf DNA LoBind tube and mix by vortexing.
AB
ReagentVolume
Proteinase K10 µl
RNase A3 µl
Total (including your sample)113 µl
Proteinase K and RNaseA treatment

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.
Add Amount200 µL of CTAB buffer and mix by vortexing.

Mix
Extractions performed using the CTAB buffer improves sequencing health and output compared to using NEB Lysis Buffer from the Monarch Spin gDNA Extraction Kit.
Expected result
IMPORTANT: The full 30-minute incubation must be completed to ensure the proteinase and RNase activity occurs, regardless of turbidity.
Duration00:30:00

30m
Incubate at Temperature56 °C for 30 minutes in a thermomixer at 1000 RPM: Shaker1000 rpm, 56°C, 00:30:00 Use Thermomixer .
Note
Depending on the bacterial input, the solution may become fully transparent or may remain hazy.

INFO: The Monarch Spin gDNA Extraction 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.

Preheat the Monarch® gDNA Elution Buffer to Temperature60 °C for later use.

Note
IMPORTANT: The ratio of binding buffer to lysate (2:1) is important for the column binding efficiency.


We recommend taking throughAmount200 µL of lysate for use with Amount400 µL of binding buffer per sample.

To run more samples from the same isolate, the volumes can be increased in relation to the 2:1 ratio of buffer to lysate.
Combine the following reagents in a clean 1.5 ml Eppendorf DNA LoBind tube:
AB
ReagentVolume
Lysate from the previous step200 µl
Monarch gDNA Binding Buffer400 µl
Total600 µl
Monarch gDNA Binding Mixture

Ensure the reaction is thoroughly mixed by pipetting (gently!) until the liquid is homogenous.
Mix
gDNA Binding and Elution
gDNA Binding and Elution
8m
8m
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.
Mix
Transfer the reaction to a Monarch® Spin Column pre-inserted into a Monarch® collection tube, without touching the upper column area.
Close the tube and centrifuge for Centrifigation1.000 x g, 20°C, 00:03:00 to bind the gDNA and then for 1 minute at maximum speed (>12,000 x g) Centrifigation16000 rpm, 20°C, 00:01:00 , 1 min at maximum speed to clear the membrane to clear the membrane.

4m
Do not empty or remove the collection tube from the centrifuge when changing settings.
Remove the gDNA spin column from the collection tube and discard the tube with the flow-through.
Transfer the gDNA spin column to a new collection tube and add Amount500 µL Monarch® gDNA Wash Buffer.

IMPORTANT: Do NOT vortex.

Pipetting
Critical
Close the collection tube and invert multiple times until the wash buffer reaches the cap.
Immediately centrifuge the collection tube for 1 minute at maximum speed (>12,000 x g). Centrifigation16000 rpm, 20°C, 00:01:00 , Maximum speed Remove the gDNA spin column from the collection tube to discard the flow-through.

1m
Re-insert the gDNA spin column into the collection tube and add Amount500 µL gDNA wash buffer and close the tube.

Immediately centrifuge the collection tube for 1 minute at maximum speed (>12,000 x g) Centrifigation16000 rpm, 20°C, 00:01:00 , Maximum speed . Remove the gDNA spin column from the collection tube to discard the flow-through.

1m
After centrifugation, ensure there is no liquid in the gDNA spin 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 Temperature60 °C is essential for maximum recovery of larger gDNA fragments.

Place the gDNA spin column in a DNase-free 1.5 ml microfuge tube and add Amount100 µL of preheated Monarch® gDNA Elution Buffer. Close the tube and incubate at room temperature for Duration00:01:00 .

TIP: If extractions are not yielding high enough concentrations of DNA forAmount200 ng per sample, less gDNA elution buffer can be run through the column to increase the concentration.
Note
Note: Elution of less than Amount100 µL results in lower total yield, and elution of less thanAmount50 µL is not recommended.


1m
Centrifuge for 1 minute at maximum speed (>12,000 x g) to elute the gDNA into the 1.5 ml microfuge tube.Centrifigation16000 rpm, 20°C, 00:01:00 , Maximum speed

1m
The gDNA spin column can be discarded.
CHECKPOINT: Quantify Amount1 µL the eluted sample using a Qubit fluorometer. Expected yield is ~15-20 ng/µl per sample with a DNA Integrity Number (DIN) of 9.

Analyze
Critical
END OF STEP: Take forwardAmount200 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. We recommend storing the DNA frozen (-30 to -15°C or -90 to -65°C). We will freeze the DNA and use it for quantification in the following lab sessions before sequencing.

Critical
Pause
Temperature
Acknowledgements
We appreciate the support from the NC State Biotechnology Program and students who have helped improve this and other protocols.