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MAG Bead DNA Extraction Protocol
Last updated date: Jul 11, 2023 Views: 730 Forks: 0
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Reagents:
- Sterile 20mg/mL Proteinase K (Invitrogen, Catalogue Number 25530-049)
- Mag Bead Kit (LGC Catalogue Number 40410)
- Extra Mag Bead Lysis buffer BL (LGC Catalogue Number NAP40011)
- Extra Mag Bead Binding buffer BL (LGC Catalogue Number NAP40101)
Equipment:
- Sterile pipette tips (previously unopened boxes only)
- Heat block (VWR, Catalogue number:12621-088)
- Vortex
- Sterile Axygen low binding eppendorf tubes (Axygen, Catalogue Number 31104081)
- Sterile screw top 2 ml tubes with O-rings (Sarstedt, 72.693.005)
- 0.3g 0.1mm zirconium beads (Biospec, Catalogue number 11079101z)
- Ice and ice bucket
- BioSpec Mini Bead Beater-24 (BioSpec, Catalogue number 112011)
- VWR Tube Rotator (VWR, Catalogue number 10136-084)
- Magnet (Permagen, Catalogue number MSR24)
- 15mL Falcon Tubes
- Benchtop centrifuge or minifuge
- 4dC Refridgeration
- Tube racks
Method:
The day before the extractions:
- Prepare the following tubes the day before the extractions.
- 4 x sterile Axygen low binding eppendorf tubes (includes 2 x for DNA aliquots)
- 1 x sterile screw top 2 ml tubes (with O-ring) containing 0.3g 0.1mm zirconium beads Label tubes with the sample identifiers.
The day of the extractions:
| Step | Procedure |
| Step 01 | Thaw CSF samples on ice |
Step 02 | Vortex thawed liquid for 20 seconds |
Step 03 | Take 100 µl of the CSF to a sterile eppendorf tube |
Step 04 | Add 20 µl of 20mg/mL Proteinase K to each extraction |
Step 05 | Incubate at 55°C for 10mins |
Step 06 | Add 250 µl of Mag Bead lysis buffer |
Step 07 | Mix all tubes by gentle vortexing for 15s |
Step 08 | Quick spin the tube, then add the mixture to the beads |
Step 09 | Bead-beat for 3 mins |
Step 10 | Place all tubes on ice for at least 1 minute |
Step 11 | Repeat the bead-beat for 3mins |
Step 12 | Place all tubes on ice for at least 1 minute |
Step 13 | Centrifuge at 10000rpm for 10 minutes |
Step 14 | Take the supernatant to a new tube |
Step 15 | Prepare 325 µl (x number of tubes +2) of bead-binding buffer and 10 µl (x number of tube +2) of mag beads in a 15mL falcon (See Table 1). |
Step 16 | Add 335 µl bead-binding buffer mixture to each tube |
Step 17 | Mix by gentle vortexing |
Step 18 | Incubate for 30 minutes at room temperature with continual mixing. |
Step 19 | Centrifuge briefly to collect fluid in the bottom of the tube |
Step 20 | Bring the magnet into contact with the tubes and incubate for 1min at RT to allow the magnetic particles to form a pellet. |
Step 21 | Remove and discard as much of the supernatant as possible, taking care not to disrupt the pellet. |
Step 22 | Remove the tubes from the magnet and resuspend the particles with 200 µl of wash buffer 1 and mix thoroughly by pipetting up and down five times or until the pellet is fully suspended. |
Step 23 | Incubate at RT for 5 mins with mixing |
Step 24 | Centrifuge briefly to collect all fluid at the bottom of the tube |
Step 25 | Bring tubes into contact with the magnet for 1 min at RT to allow the particle pellet to form. |
Step 26 | Remove the supernatant and discard. Ensure as much of the supernatant is removed as possible without dislodging the particle pellet. |
Step 27 | Repeat steps 19-26 with wash buffer 2. |
Step 28 | Dry the tubes (with the lid open) for 5 mins at 55°C. |
Step 29 | Add 63 µl of Elution Buffer BL and resuspend the pellet. Mix thoroughly by setting a pipette to 50uL and pipetting up and down 5 times or until the pellet is fully resuspended. |
Step 30 | Incubate at 55°C for 10mins, agitating the sample during the time period. Use a heated shaker or vortex periodically. |
Step 31 | Incubate at 4°C for 5mins |
Step 32 | Centrifuge briefly to collect all fluid at the bottom of the tube |
Step 33 | Bring magnet into contact with the sample tubes. Wait for 3 minutes at RT to allow the mag particles to form a pellet. |
Step 34 | Remove the eluate (contains the DNA) to a new tube. To avoid particle transfer it is recommended that only 54 µl of the eluate is transferred (2x27 µl aliquots). |
Step 35 | Aliquot the DNA into 2 tubes - 1 for sequencing, and 1 for qPCR and DNA conc. |
Table 1: Aliquot amounts required for each solution.
| Component | For 1 tube | for 14 tubes |
| Prot-K | 20 µl | 280 µl |
| Lysis Buffer | 250 µl | 3500 µl |
| Binding Buffer | 325 µl | 4550 µl |
| Mag Beads | 10 µl | 140 µl |
| Wash Buffer 1 | 200 µl | 2800 µl |
| Wash Buffer 2 | 200 µl | 2800 µl |
| Elution Buffer | 54 µl | 756 µl |
References:
Biesbroek, G. et al. Deep sequencing analyses of low density microbial communities: working at the boundary of accurate microbiota detection. PLoS ONE 2012 7: e32942.
Copyright: © 2023 The Author(s); This is an open-access article under the CC BY license (https://creativecommons.org/licenses/by/4.0/).
How to cite：
Readers should cite both the Bio-protocol preprint and the original research article where this protocol was used:
- Simon, T and Pope, C(2023). MAG Bead DNA Extraction Protocol. Bio-protocol Preprint. bio-protocol.org/prep2364.
- Whitlock, K. B., Pope, C. E., Hodor, P., Hoffman, L. R., Jr., D. L. L., McDonald, P. J., Hauptman, J. S., Ojemann, J. G. and Simon, T. D.(2021). Characterization of cerebrospinal fluid (CSF) microbiota from patients with CSF shunt infection and reinfection using high throughput sequencing of 16S ribosomal RNAgenes. PLoS ONE 16(1). DOI: 10.1371/journal.pone.0244643
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