Dried Blood Spot Extraction (Optional)

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This step prepares a dried blood spot extraction using the Bead-Linked Transposomes (BLT) to tagment DNA, which is a process that fragments and tags the DNA with adapter sequences.

The Flex Lysis kit contains 800 µl BLB. Preparing 96 samples following the Blood Lysis workflow requires 672 µl (96 samples x 7 µl = 672 µl).

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. A fully lysed sample makes sure that any blood borne pathogens are eliminated and the sample is no longer biohazardous.

• BLB (Blood Lysis Buffer)
• PK1 (Proteinase K)
• IPB(Illumina Purification Beads)
– 30 ml
– 100 ml
– 400 ml
• Dried Blood Spots (DBS) card (GE Healthcare, catalog # 10534320)
• EtOH (Freshly prepared 80% ethanol)
• Nuclease-free water
• 96-well PCR plate
• 1.5 ml tube
• [Optional] Microseal 'B' adhesive seal
• BLB (Blood Lysis Buffer) is shipped frozen but stored at room temperature. Keep at room temperature for optimal use.
• Choose the appropriate IPB kit for your sample size.
1. Prepare the following consumables:
• BLB—Check for any precipitates. If present, heat at 37°C for 10 minutes and vortex to resuspend.
• DBS—Add EDTA-stablized blood (70 μl per shot) or add a finger-poke sample.
• PK1—Place on ice.
• IPB—Keep at room temperature.
2. Preheat the thermal mixer to 56°C.
1. Prepare 5 × 3 mm2 punches from a DBS card and add them to a 1.5 ml tube.
2. Combine the following reagents per reaction to create a lysis master mix.
• BLB (20 μl)
• PK1 (2 μl)
• Nuclease-free water (178 μl)
3. Vortex and centrifuge the lysis master mix briefly.
4. Add 200 μl master mix to each sample.
5. Pipette to mix.
6. Shake on the preheated thermal mixer at 56°C for ten minutes.
7. Briefly centrifuge the sample.
8. Without removing the punches, transfer all supernatant (~190 μl) to a new 1.5 ml tube.
9. Vortex and invert IPB multiple times until resuspended.
10. Add 90 μl of IPB to the lysed sample.
11. Using a pipette set to 200 μl, thoroughly mix IPB and sample.
12. Incubate at room temperature for five minutes.
13. Place on the magnetic stand and incubate for five minutes.

The lysis reaction turns brown, so the IPB are not visible.

14. Without disturbing the bead pellet, pipette to remove the supernatant.
15. Make sure that a bead pellet is at the bottom of the tube before discarding the supernatant.
16. If the beads are accidentally aspirated:
a. Return the sample to the tube and allow it to settle.
b. Remove and discard the supernatant.
17. Wash beads as folows.
a. Keep on the magnetic stand and add 200 µl fresh 80% EtOH to each tube.
b. Incubate on the magnetic stand and wait 30 seconds.
c. Remove and discard all supernatant from each tube.
18. Wash beads a second time.
19. Wash beads a third time.
20. Remove all residual EtOH from each tube.
21. Remove and discard any residual EtOH.
22. Air-dry on the magnetic stand for five minutes.
23. Remove from the magnetic stand.
24. Resuspend IPB in 30 μl of nuclease-free water. Pipette to resuspend.
25. Transfer the resuspended beads to a 96-well PCR plate.
26. If you are not stopping, proceed immediately to step Step 3 of Tagment Genomic DNA.

SAFE STOPPING POINT

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