Blood Lysis (Optional)

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Use this protocol when performing the Illumina DNA Prep with Exome 2.5 Enrichment workflow using blood sample inputs with the Flex Lysis Reagent Kit. This protocol has been validated using fresh whole blood collected in EDTA collection tubes. Store the blood at 4°C and process it within 3 days. The use of frozen blood has not been validated and cannot be recommended.

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

Blood is a potential source of infectious diseases. Follow site-specific procedures to ensure the safe handling of blood samples. During the lysis protocol, make sure that the entire blood sample is fully lysed (brown in color following the heat incubation step) before proceeding to subsequent steps. This will make sure that any bloodborne pathogens are eliminated and the sample is no longer biohazardous.

• BLB (Blood Lysis Buffer)
• IPB (Illumina Purification Beads)
• PK1 (Proteinase K)
• Freshly prepared 80% ethanol (EtOH)
• Nuclease-free water
• 96-well PCR plate
• EDTA collection tubes (for blood sample collection)
• 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

BLB

15°C to 30°C

If frozen, thaw at room temperature. If precipitates are observed, heat at 37°C for 10 minutes and vortex until resuspended.

IPB

15°C to 30°C

Vortex and invert to mix.

PK1

‑25°C to ‑15°C

Place on ice until needed.
2. Prepare 150 µl fresh 80% EtOH from absolute ethanol. Include a 20% overage.
3. Save the following BLP program on the thermal cycler:
• Choose the preheat lid option and set to 100°C
• Set the reaction volume to 70 µl
• 56°C for 10 minutes
1. Combine the following volumes to prepare the Lysis Master Mix. Multiply each volume by the number of samples being processed.
• BLB (8.4 µl)
• PK1 (2.4 µl)
• Nuclease-free water (37.2 µl)

Reagent overage is included in the volume to ensure accurate pipetting.

2. Vortex and centrifuge the Lysis Master Mix.
3. Invert the EDTA collection tube 10 times to mix.
4. Transfer 10 µl blood from the tube to one well of a 96-well PCR plate.
5. Add 40 µl Lysis Master Mix to each sample.
6. Vortex and invert IPB multiple times to resuspend.
7. Add 20 µl IPB to the well.
8. Using a pipette set to 50 µl, slowly pipette 10 times to mix, and then seal.
9. Place on the preprogrammed thermal cycler and run the BLP program.
10. Place on a magnetic stand and wait 5 minutes.

The dark brown color of the blood from the lysis reaction will keep the liquid from becoming clear. The beads migrate after 5 minutes.

11. Without disturbing the beads, remove and discard supernatant.
12. 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).
13. Add 150 µl fresh 80% EtOH to the well.
14. Incubate on the magnetic stand for 30 seconds.
15. Pipette to remove and discard the 80% EtOH.
16. Use a 20 µl pipette to remove and discard all residual EtOH.
17. Remove the plate from the magnetic stand.
18. Add 32 µl nuclease-free water and pipette to resuspend.
19. Seal the plate.
20. Centrifuge plate at 280 × g for 30 seconds.
21. Remove seal, place on magnetic stand and wait until the liquid is clear (~2 minutes).
22. Transfer 30 µl supernatant to a new 96-well PCR plate.
23. 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 a Microseal 'B' and store at 2°C to 8°C for up to 3 days.