Saliva Lysis (Optional)

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Use this protocol when performing the Illumina DNA Prep workflow using saliva sample inputs. This protocol is validated for saliva collected only in Oragene DNA saliva collection tubes. The saliva is mixed with the Oragene Dx solution contained in the collection tube, making it stable at room temperature.

This protocol is expected to generate > 100 ng of DNA output at the end of the saliva lysis step.

Saliva is a potential source of infectious diseases. Follow site-specific procedures to ensure the safe handling of saliva samples.

• IPB (Illumina Purification Beads)
• 96-well PCR plate
• EtOH (Freshly prepared 80% ethanol)
• Nuclease-free water
• Oragene DNA collection tubes (for saliva sample collection)
• IPB
– Must be at room temperature before use.
– Vortex before each use.
– Vortex frequently to make sure the beads are evenly distributed.
– Aspirate and dispense slowly due to the viscosity of the solution.
1. Prepare the following consumables.

Item

Storage

Instructions

IPB

Room temperature*

Vortex and invert to mix.

Saliva samples in Oragene DNA collection tubes

Room temperature

For information on sample preparation and storage, refer to the DNA Genotek website.

*IPB is included in the Illumina DNA Prep Kit.

2. Prepare fresh 80% EtOH from absolute ethanol.
1. For each sample, add 20 µl nuclease-free water to one well of a 96-well PCR plate.
2. Vortex the heat-treated Oragene DNA collection tube.
3. Transfer 30 µl saliva sample from the tube to the well containing nuclease-free water.
4. Slowly pipette to mix.

For viscous samples, use a wide-bore pipette tip for more accurate pipetting.

5. Vortex and invert IPB multiple times to resuspend.
6. Add 20 µl IPB to the well.
7. Using a pipette set to 50 µl, slowly pipette 10 times to mix.
8. Incubate at room temperature for 5 minutes.
9. Place on a magnetic stand and wait 5 minutes.
10. Without disturbing the beads, remove and discard supernatant.
11. If beads are aspirated into pipette tips, dispense back to the plate on the magnetic stand, and wait until the liquid is clear (~2 minutes).
12. Add 150 µl fresh 80% EtOH to the well.
13. Incubate on the magnetic stand for 30 seconds.
14. Use a 20 µl pipette to remove and discard all residual EtOH.
15. Remove the plate from the magnetic stand.
16. Add 30 µl nuclease-free water and pipette to resuspend.
17. If you are not stopping, proceed immediately to step Step 3 of Tagment Genomic DNA.

SAFE STOPPING POINT

Seal the plate with a Microseal 'B' adhesive seal and store at 2°C to 8°C for up to 3 days.