Colony-Based DNA Extraction
- One input option for this kit is DNA from colony-based extraction. This uses some of the Flex reagents and is an easy way to go from a colony directly to library preparation. It is included in the protocol here, but any standard DNA extraction technique may be used.
- This has been adapted from the Nextera DNA Flex Microbial Colony Extraction found here.
Reagents and Equipment:
- Illumina Purification Beads (IPB) (Included with Nextera FLEX kit) or AMPure XP Beads
- PowerBead tubes, glass, 0.5 mm (QIAGEN, catalog #13116-50)
- Disposable inoculation loop, 10 μl (Fisher,catalog#12870155)
- 1.5 mL microcentrifuge tubes
- Freshly made 80% ethanol
- Nuclease-free water
- Magnetic Stand
- Vortex-Genie 2 with vortex adapter for 1.5 mL tubes (24)
Extraction
Remove SPB or Ampure Beads from the refrigerator and allow to stand for 30 minutes at room temperature.
Add 300 ul of nuclease-free water to the PowerBead tubes containing 0.5 mm glass beads.
Using a 10 μl disposable inoculation loop, pick up to a half loopful of colonies from the bacterial culture plate.
Resuspend colonies in the PowerBead tube containing glass beads and nuclease-free water.
Fit a Vortex-Genie 2 with a vortex adapter.
Vortex at speed 6 for five minutes. For environmentally-resilient bacteria, vortex at max speed for 10 minutes
Centrifuge at 20,000 × g for two minutes. Make sure that the glass beads are at the bottom of the PowerBead tube.
Transfer all supernatant (~150 μl) without the glass beads to a new 1.5 ml tube.
Transfer 50 μl of this supernatant to a new 1.5 ml tube.
The cultures we work with have very small colonies, and so we haven’t been able to test this protocol using entire half-loopfuls of bacteria. Because of the low input, we do not dilute the supernatant. However, the official protocol recommends transferring 20 μl of supernatant into a 1.5 mL tube containing 30 μl of nuclease-free water. If an entire half-loopful of colonies was picked, you may have more success by diluting your supernatant.
Vortex and invert beads several times to resuspend
Add 20 μl beads to the 1.5 ml sample tube containing 50 μl supernatant.
Using a pipette set to 50 μl, mix ten times to thoroughly mix the beads and sample.
Incubate at room temperature for five minutes.
Place on the magnet until the beads have fully migrated to the side of the tube (~5 minutes).
Remove the supernatant without disrupting the bead pellet.
Make sure that a bead pellet is at the bottom of the tube before discarding the supernatant.
If the beads are accidentally aspirated, return the sample to the tube and allow it to settle, then remove and discard the supernatant
Wash two times as follows:
- Add 200 μl fresh 80% EtOH to each tube.
- Incubate on the magnetic stand for 30 seconds.
- Remove and discard all supernatant from each tube.
Remove any residual EtOH using a P20 pipette.
Air-dry the tubes on the magnetic stand (~10 minutes).
Remove the tubes from the magnetic stand.
Resuspend beads in 32.5 μl of RSB. Pipette to mix.
Incubate at room temperature for 2 minutes.
Place on a magnetic stand and wait until the liquid is clear (~2 minutes).
Transfer 30 μl to a new 1.5 ml tube.
We average ~10 ng/ul of C. difficile using this protocol.
You can now proceed to the Tagment DNA Step for Standard Nextera Flex.