Nov 24, 2025

Public workspaceDNA Extraction from inoculated broth for metagenomics using the Promega Maxwell RSC cultured kit V.2

Version 1 is forked from DNA Extraction from Inoculated Broth for WGS using the Maxwell RSC Cultured Cells DNA Kit
DOI
dx.doi.org/10.17504/protocols.io.6qpvrqb72lmk/v2
DNA Extraction from inoculated broth for metagenomics using the Promega Maxwell RSC cultured kit
  • Christopher Duda1,
  • Anna Brover1,
  • Padmini Ramachandran1,
  • Christopher Grim1
  • 1FDA
  • GenomeTrakr
    Tech. support email: genomeTrakr@fda.hhs.gov
Christopher Duda
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DOI: https://dx.doi.org/10.17504/protocols.io.6qpvrqb72lmk/v2
Protocol Citation: Christopher Duda, Anna Brover, Padmini Ramachandran, Christopher Grim 2025. DNA Extraction from inoculated broth for metagenomics using the Promega Maxwell RSC cultured kit. protocols.io https://dx.doi.org/10.17504/protocols.io.6qpvrqb72lmk/v2Version created by Christopher Duda
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: November 24, 2025
Last Modified: November 24, 2025
Protocol Integer ID: 233380
Keywords: DNA Extraction, Automation, broth for metagenomic, metagenomic, extracting dna, metagenomic enrichment, dna extraction, dna from gram, promega maxwell rsc instrument, promega maxwell rsc, selective enrichment broth, positive bacterial cells from pure culture, genomic dna, universal enrichment broth, maxwell rsc, maxwell rsc 48 instrument, dna, inoculated broth, positive bacterial cell, extraction method purify, extraction, purification process, information for extraction
Abstract
The Maxwell® RSC Cultured cells DNA Kit is used with the Maxwell RSC or Maxwell RSC 48 Instrument from Promega. This extraction method purifies genomic DNA (gDNA) from pure culture and/or from metagenomic enrichments efficiently using automation. The kit utilizes a silica-based paramagnetic particle that is used during the purification process.

This protocol will provide information for extraction from universal enrichment broth or selective enrichment broth. Additional steps are performed when extracting DNA from gram-positive organisms.

This SOP is applicable to all laboratories with the intent of obtaining high quality, purified genomic DNA or quasi-metagenomic DNA through the use of a Promega Maxwell RSC Instrument. DNA can be isolated from both gram-negative or gram-positive bacterial cells from pure culture or enrichments for use in subsequent sequencing protocols.
Guidelines






Materials
Reagents:
Maxwell® RSC Cultured Cells DNA Kit -- Cat# AS1620 [Promega]
DNase free, RNase free, molecular grade water -- Cat# 10977015 [Invitrogen] or equivalent
PureLink™ RNase A (20 mg/ml) -- Cat# 12091021 [ThermoFisher Scientific] or equivalent
TE, pH 7.0, RNase-free -- Cat# AM9861 [ThermoFisher Scientific] or equivalent
Lysozyme -- Cat# L4919 [Sigma Aldrich] or equivalent
Metapolyzyme https://www.sigmaaldrich.com/US/en/product/sigma/mac4l?srsltid=AfmBOopn8iMZxurbqDXFPbwvM9WQfM9RHM5qPgtAHS0Acn0EvNobDGT2 for metagenomic DNA extraction from enrichments

Equipment:
Maxwell® RSC or Maxwell® RSC 48 Instrument
Pipettors
Centrifuge
Incubator
Heatblock
Vortexer

Consumables:
2.0 ml Eppendorf Safe-Lock Tubes -- Cat# 05-402-7 [ThermoFisher Scientific]
1.5 ml Eppendorf DNA LoBind Microcentrifuge Tubes -- Cat# 13-698-791 [ThermoFisher Scientific]
Pipette Tips

Troubleshooting
Safety warnings




Refer to the full SDS for detailed safety information.
Download AS1620-en-US.pdfAS1620-en-US.pdf369KB

Pre-processing of Culture Enrichments for Gram Positive Communities
Due to the thick peptidoglycan layer of Gram-Positive bacteria extra lysing is necessary. Follow the steps below for this pre-processing. While we show 2 different lysis methods, *DO ONLY step 3 OR 4*
1. Spin enrichment cultures down at 3,000 rcf for 20 minutes.
2. Remove and discard the supernatant from the tube.
3. Continue onto EITHER Step 3 or 4.
Lysis With LE-Lysozyme *Only do one lysis step*
  1. Resuspend the pellet with 400 μl of the TE-Lysozyme buffer.
  2. Vortex for 5-10 seconds
  3. Incubate at 37 ºC for at least 30 minutes. If the enrichment contains a highly gram-positive community, an increased lysis time may be necessary.
  4. Add 400 μL of the lysate to the first well of the maxwell kit cartridge.
  5. Proceed to Cultured Cells DNA Cartridge Preparation
Lysis With Metapolyzyme *Only do one lysis step*
  1. Resuspend the pellet with 50ul of metapolyzyme (Sigma-aldrich).
  2. Vortex for 5-10 seconds
  3. Incubate at 37 ºC for at least 30 minutes. If the enrichment contains a highly gram-positive community, an increased lysis time may be necessary.
  4. Add 400 μL of the lysate to the first well of the maxwell kit cartridge.
  5. Proceed to Cultured Cells DNA Cartridge Preparation
Loading Enrichments into Maxwell Cartridges
Transfer at least 8 mL of Rappaport-Vassiliadis Broth (RV) or Tetrathionate broth (TT) cultures or overnight general enrichment (mBPW, UPB, TSB, APW) to a 15 mL centrifuge tube
1. For RV and/or general enrichment, spin cultures down at 3,000 rcf for 20 minutes.
2. Remove and discard the supernatant from the tube.
3. Using a Maxwell Promega DNA isolation kit cartridge, remove 400 μL of the lysis buffer (well 1, see step 4 for details) and resuspend cells by vortexing.
5. Add the lysis buffer and cell solution back to the first well of the maxwell kit cartridge.
1. For TT, Spin cultures down at 3,000 rcf for 20 minutes
2. Remove and discard the supernatant from the tube.
3. Using a Maxwell Promega DNA isolation kit cartridge, remove 400 μL of the lysis buffer (well 1, See step 4 for details) and resuspend by vortexing.
5. Spin down tubes at 300 rcf for 3 minutes to remove solid salts from the supernatant.
6. Add the lysis buffer and cell solution back to the first well of the maxwell kit cartridge.
Cultured Cells DNA Kit Cartridge Preparation
1. Turn on the instrument and associated PC. Start the RSC software and wait for the instrument to initialize.

2. Open the door of the instrument by pressing the Door icon at the top right corner of the software.

3. Remove the deck tray(s) from the instrument. Press the Door icon to close the instrument door.

4. Place the number of cartridges needed for the number of samples into the deck tray. The cartridges are inserted with Well 1 (lysis buffer) at the top of the tray. Figure 1 illustrates the well locations on the cartridge. Press down on the cartridge to snap it into position.

5. Carefully peel back the seal on the cartridges. Ensure that the entire seal is removed.

6. Add a plunger to Well 8 in each cartridge.


Figure 1. Cartridge setup

7. Place elution tubes (provided with kit) into the elution tube position with the lid facing the bottom of the deck tray (Figure 2). Add 100 μl of Elution buffer to each tube. Ensure there are no bubbles in the tubes, and the buffer is at the bottom of each tube.

Figure 2. Elution Buffer Tube and Lid positioning
8. Transfer 400 μl of the sample culture to the lysis buffer in Well 1 of the corresponding cartridge and pipette gently up and down 10 times to mix. Proceed to the Instrument Setup and Extraction Run step.

9. Optional: RNase A Treatment
a. Allow cells to lyse for 10 minutes in Well 1.
b. Add 2 μl of RNase A to each sample in Well 1. Pipette up and down to mix.
c. Incubate for an additional 10 minutes prior to starting the instrument.



Instrument Setup and Extraction Run
1. In the RSC Software, press Start to access the extraction method selection screen.

2. Select Cultured Cells DNA method, then press Proceed.

3. Select the lanes being used by touching them. Sample names can be inputted at this time.
Note: For the RSC48, press the "BACK" and "FRONT" buttons on the bottom of the screen to move to each respective rack.

4. Press Proceed to continue. The instrument door will open.

5. Install the deck tray(s) into the platform by angling the back of the tray and inserting first. Press down the front of the tray until it snaps into place. Ensure the deck tray is level and fully seated.
Note: For the RSC48, ensure the tray labelled FRONT and BACK are placed in the correct places in the instrument.

6. Verify that all of the cartridges are properly inserted into the tray, the elution tubes are present and uncapped, and then plungers are in Well 8.
Note: Ensure there are no old plungers left on the instrument from a prior run.

7. Press Start to begin the extraction run.

8. Follow on-screen instructions at the end of the run.

9. Remove the deck tray(s) from the instrument. The DNA can be transferred to 1.5 ml LoBind Tubes for storage.

10. Remove the cartridges and plungers from the deck trays and discard in a biohazard container.

11. Clean the tray with 70% Ethanol. Do NOT use bleach to clean the trays as this reacts with Guanidine Thiocyanate.

12. Proceed to the Cleaning and Decontamination steps.

13. Proceed to quantify the DNA using the DNA Quantification using the Qubit Fluorometer SOP.
Cleaning and Decontamination
The Maxwell RSC instrument should be wiped down with 70% Ethanol as needed.

Follow up each extraction with the Sterilization process on the instrument.