Jun 03, 2026
2. Shearing of HMW Bacterial DNA Using the MP Biomedicals™ FastPrep-96™ High-throughput Bead Beating Grinder and Lysis System
- 1CFSAN/FDA;
- 2USDA;
- 3US FDA
- GenomeTrakrTech. support email: [email protected]
Protocol Citation: Jae Hee Jang, Ellie Meeks, Kathryn Judy, Maria Hoffmann 2026. 2. Shearing of HMW Bacterial DNA Using the MP Biomedicals™ FastPrep-96™ High-throughput Bead Beating Grinder and Lysis System. protocols.io https://dx.doi.org/10.17504/protocols.io.q26g7y28kgwz/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: May 02, 2023
Last Modified: June 03, 2026
Protocol Integer ID: 81307
Keywords: HMW DNA, DNA shearing, fastprep, fastprep-96, shearing of hmw bacterial dna, hmw bacterial dna extraction, shearing bacterial genomic dna, hmw bacterial dna, monarch hmw dna extraction kit for tissue, monarch hmw dna extraction kit, bacterial genomic dna, hmw dna, protocol results in dna fragment size, dna fragment size, use dna, throughput bead beating grinder, mp biomedical, shearing
Abstract
This protocol details the process of shearing bacterial genomic DNA using the MP Biomedicals™ FastPrep-96™ High-throughput Bead Beating Grinder and Lysis System. Using this protocol results in DNA fragment sizes of about 18kb, a suitable input size for the "Post-Shearing 1x SMRTbell Bead Clean Up" protocol. Use DNA obtained from the "HMW Bacterial DNA Extraction Using NEB Monarch® HMW DNA Extraction Kit for Tissue" protocol for optimal results.
Guidelines
- When transferring samples, make sure to use wide-bore 200 µl pipette tips to avoid unwanted shearing.
- When placing samples in the 96 deep well plate, avoid placing samples in adjacent or diagonal wells to minimize chances of cross contamination.
- Be careful when removing the adhesive plate sealing film; remove it gently to avoid splashing of samples.
Materials
Materials Required but Not Supplied:
- Microcentrifuge
- Thermal mixer containing a 1.5 ml tube block (optional: 2 ml tube block for elution)
- Recommended: vertical rotating mixer (e.g., Thermo Scientific‱ HulaMixer‱ Sample Mixer).
- Ethanol (≥ 95%).
- Cold PBS, 300 µl per sample (Low Input: 150 µl per sample). Alternatively, TE or Tris buffer can be used.
- Isopropanol, 550 µl per sample (Low Input: 275 µl/sample).
- 1.5 ml DNase-free, low DNA binding microfuge tubes (e.g., Eppendorf‱ DNA LoBind‱, #0030108051) are recommended for elution and storage (1 per sample); it is especially important to use low DNA binding tubes if working with UHMW DNA, which tends to bind to plastic surfaces.
- For Gram-negative bacteria: Lysozyme (25 mg/ml, 10 µl per sample)
- For Gram-positive bacteria: STET Buffer (Current Protocols in Molecular Biology) containing Lysozyme (10 mg/ml) can be an effective lysis agent (150 µl or 300 µl per sample).
- Additional lysis agents may be required (e.g., lysostaphin).
- MP Biomedicals™ FastPrep-96™ High-throughput Bead Beating Grinder and Lysis System
- Low TE buffer (PacBio SMRTbell prep kit 3.0, #102-178-400)
- 96 DeepWell Plate 1mL (Cat. No. 12566120). 24 samples max per plate.
- Microseal 'B' PCR Plate Sealing Film, adhesive, optical#MSB1001.
- Pipettes and standard pipette tips.
- Wide-bore 200 µl pipette tips.
- 96-well V-bottom plates (Perkin Elmer).
- 0.2 mL PCR tubes.
Safety warnings
Safety information
Noise Warning: The FastPrep-96‱ High-throughput Bead Beating Grinder and Lysis System emits a loud noise (Maximum sound level: <70 dB) that is alarming. It is important to alert people in the vicinity of the machine before usage. Earplugs or leaving the room is recommended for the duration of the machine’s run.
Before start
- Review the complete protocol before beginning.
- Quantify samples and calculate amount of DNA/TE buffer needed to reach 1000 ng in 50 µl.
Dilution of DNA
20m
In 1.5 mL Eppendorf tubes, dilute samples so that each has 1000 ng of DNA in 50 µL . Dilute using appropriate amounts of low TE buffer.
- Alternatively, samples may be diluted directly in a 96 deep well plate.
Transfer diluted samples to two 96 deep well plates.
- Use wide bore pipette tips when transferring samples to avoid unwanted shearing.
- Space out samples to avoid cross contamination; do not place samples in adjacent or diagonal wells.
- Split samples across two plates evenly (e.g. 32 samples would be split into 16 samples per plate). Plates should be as equal in mass as possible.
- Center samples on each plate.
- Two plates are always required. If shearing only 1 sample, use water to fill the corresponding well on a second plate to act as a balance.
Figure 1: An example layout of 24 samples on a plate. The maximum number of samples per run is 48 (each run can support two plates).
Cover plate with an adhesive plate sealing film.
- Make sure that the film is sealed around the entire circumference of the wells containing samples.
Shearing of DNA
5m
Insert deep well plates into the FastPrep-96™ High-throughput Bead Beating Grinder and Lysis System.
Figure 2: The FastPrep-96™ High-throughput Bead Beating Grinder and Lysis System
Turn on the FastPrep using switch near the lower right corner of the back of the machine.
Set the FastPrep to the proper settings.
- Use the dials on either side to adjust the time and speed settings.
- 1600 rpm for 00:00:22 for fragment sizes around 18kb.
- Increase rpm or time for shorter fragments; decrease rpm or time for longer fragments.
- Going below 1600 rpm is not recommended if more uniform fragment sizes are desired.
Figure 3: The FastPrep interface
22s
Insert two deep well plates into the FastPrep.
- Access the plate compartment by lifting the clear outermost lid (Figure 4), loosening the screw that secures the blue inner lid (Figure 5), and lifting the blue inner lid (Figure 6).
- Insert the two plates, one on each side of the plate compartment.
- Replace the inner lid and tighten the screw until you hear a click.
- Close the outermost lid.
Figure 4: Lifting the clear outermost cover.
Figure 5: Loosening the screw to access the plate compartment. Turn counterclockwise to loosen, and clockwise to tighten.
Figure 6: Lifting the blue inner lid to access the plate compartment.
Press the green "START" button to run the plates at 1600 rpm for 00:00:22 for fragment sizes around 18kb.
- Increase rpm or time for shorter fragments; decrease rpm or time for longer fragments.
- Going below 1600 rpm is not recommended if more uniform fragment sizes are desired.
22s
Take plates out of the FastPrep-96™ machine and spin them down at 2000 rpm for 00:00:30 .
30s
Transfer samples out of 96 deep well plate using wide bore pipette tips to avoid unwanted shearing.
- Transfer samples to 200 µl PCR tubes for manual cleanup using the