Saliva Lysis (Optional)

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Use this protocol when performing the Illumina DNA Prep with Exome 2.5 Enrichment 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.

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

• IPB (Illumina Purification Beads)
• Freshly prepared 80% ethanol (EtOH)
• Nuclease-free water
• 96-well PCR plate
• Oragene DNA collection tubes (for saliva sample collection)

About Reagents

• IPB
– Must be at room temperature before use
– Vortex before each use
– Vortex frequently to make sure that 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

15°C to 30°C

Vortex and invert to mix.

Saliva samples in Oragene DNA collection tubes

Room temperature

Any time after sample collection, incubate for a minimum of 1 hour at 50°C in a water bath or an air incubator (as recommended by DNA Genotek) to lyse the cells. Following heat treatment, store at room temperature. For information on long-term storage of Oragene/saliva samples at room temperature and guarantees, refer to the DNA Genotek website.
2. For each sample, prepare 150 µl fresh 80% EtOH from absolute ethanol. Include a 20% overage.
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 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 all 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 32 µl nuclease-free water and pipette to resuspend.
17. Seal the plate and centrifuge the plate at 280 × g for 30 seconds.
18. Remove seal, place on a magnetic stand and wait until the liquid is clear (~2 minutes).
19. Transfer 30 µl supernatant to a new 96-well PCR plate.
20. If you are not stopping, proceed immediately to step 3 of Tagment Genomic DNA.

SAFE STOPPING POINT

If you are stopping, seal the plate with Microseal 'B' and store at 2°C to 8°C for up to 3 days.