5. Cycle on a thermocycler using the parameters in Table 2 and
repeat Table 2 steps 2–4 for 12–18 cycles (see Note 23).
6. Add 90μL of homogenous AMPure XP beads (see Note 9) to
each Post-Index Amplified sample tube (~50μL), mix by pipetting until the solution becomes homogenous, and incubate at
room temperature for 5 min.
7. Place strip tubes on DynaMag-96 Side magnet and allow the
solution to clear (3–5 min).
8. Keeping the tubes on the magnetic stand, carefully remove the
cleared supernatant without disturbing the beads.
9. With the tubes still in place on the magnetic stand, add 200μL
of 70% ethanol and incubate for 1 min or when solution
becomes clear. Remove ethanol and wash and repeat.
10. After second wash step use a P10 tip to remove any residual
ethanol from the bottom of the tube.
11. Dry samples on thermocycler at 37 ˚C for not more than 5 min
(see Note 10).
12. Add 32μL of nuclease-free water directly to each bead pellet.
Mix by vortexing and briefly spin tube to collect contents.
Incubate for 2 min at room temperature.
13. Put tubes on magnetic rack, leave for 2–3 min to clear and
collect the supernatant (~30μL), and transfer to new 8-tube
strip.
14. Add 3μL of D1000 Sample Buffer and 1μL of D1000 ladder to
tube 1 in a new 8-tube strip (see Note 11).
15. Add 3μL of D1000 Sample Buffer and 1μL of each sample to
tubes 2–16 using 8-tube strips (see Note 12), add caps and
vortex, spin down for 1 min, and carefully remove caps.
16. Load samples into TapeStation and run D1000 tape. Assess
quality and quantity, recording the peak height (see Note 24).
Table 2
Addition of index tags by posthybridization amplification
Step
Temperature (
C)
Time
1. Initial denaturing
98
2 min
2. Denaturing
98
30 s
3. Annealing
57
30 s
4. Extension
72
1 min
5. Final extension
72
10 min
6. Cool down
4
Hold
474
Amanda Claire Brown
repeat Table 2 steps 2–4 for 12–18 cycles (see Note 23).
6. Add 90μL of homogenous AMPure XP beads (see Note 9) to
each Post-Index Amplified sample tube (~50μL), mix by pipetting until the solution becomes homogenous, and incubate at
room temperature for 5 min.
7. Place strip tubes on DynaMag-96 Side magnet and allow the
solution to clear (3–5 min).
8. Keeping the tubes on the magnetic stand, carefully remove the
cleared supernatant without disturbing the beads.
9. With the tubes still in place on the magnetic stand, add 200μL
of 70% ethanol and incubate for 1 min or when solution
becomes clear. Remove ethanol and wash and repeat.
10. After second wash step use a P10 tip to remove any residual
ethanol from the bottom of the tube.
11. Dry samples on thermocycler at 37 ˚C for not more than 5 min
(see Note 10).
12. Add 32μL of nuclease-free water directly to each bead pellet.
Mix by vortexing and briefly spin tube to collect contents.
Incubate for 2 min at room temperature.
13. Put tubes on magnetic rack, leave for 2–3 min to clear and
collect the supernatant (~30μL), and transfer to new 8-tube
strip.
14. Add 3μL of D1000 Sample Buffer and 1μL of D1000 ladder to
tube 1 in a new 8-tube strip (see Note 11).
15. Add 3μL of D1000 Sample Buffer and 1μL of each sample to
tubes 2–16 using 8-tube strips (see Note 12), add caps and
vortex, spin down for 1 min, and carefully remove caps.
16. Load samples into TapeStation and run D1000 tape. Assess
quality and quantity, recording the peak height (see Note 24).
Table 2
Addition of index tags by posthybridization amplification
Step
Temperature (
C)
Time
1. Initial denaturing
98
2 min
2. Denaturing
98
30 s
3. Annealing
57
30 s
4. Extension
72
1 min
5. Final extension
72
10 min
6. Cool down
4
Hold
474
Amanda Claire Brown