13. Prepare A-Tailing Mix for each sample, on ice.
14. Add 20μL of A-Tailing Mix to each 30μL End Repaired Sample
in 8-strip tube, mix by gentle pipetting, and close strip caps.
15. Incubate on a thermocycler at 37
C for 30 min; do not use a
heated lid.
16. Add 90μL of homogenous AMPure XP beads (see Note 9) to
each A-Tailed sample tube (~50μL), mix by pipetting until the
solution becomes homogenous, and incubate at room temperature for 5 min.
17. Place strip tubes on DynaMag-96 Side magnet and allow the
solution to clear (3–5 min).
18. Keeping the tubes on the magnetic stand, carefully remove the
cleared supernatant without disturbing the beads.
19. With the tubes still in place on the magnetic stand, add 200μL
of 70% ethanol and incubate for 1 min or when solution clears.
Remove ethanol and wash and repeat.
20. After second wash step use a P10 tip to remove any residual
ethanol from the bottom of the tubes.
21. Dry samples on thermocycler at 37 ˚C for not more than 5 min
(see Note 10).
22. Add 15μL of nuclease-free water directly to bead pellet. Mix by
vortexing and briefly spin tubes to collect contents. Incubate
for 2 min at room temperature.
23. Put tubes on magnetic rack, leave for 2–3 min to clear, collect
the supernatant (~13μL), and transfer to a new 8-strip tube.
24. Proceed immediately to adaptor ligation step.
25. Prepare Adaptor Ligation Mix for each sample, on ice.
26. Add 37μL of Adaptor Ligation Mix to each 13μL A-Tailed
Sample in an 8-tube strip, mix by gentle pipetting, and close
strip caps.
27. Incubate on a thermocycler at 20 ˚C for 15 min and do not use
a heated lid.
28. Add 90μL of homogenous AMPure XP beads (see Note 9) to
each Adapter Ligated sample tube (~50μL), mix by pipetting
until the solution becomes homogenous, and incubate at room
temperature for 5 min.
29. Place strip tubes on DynaMag-96 Side magnet and allow the
solution to clear (3–5 min).
30. Keeping the tubes on the magnetic stand and carefully remove
the cleared supernatant without disturbing the beads.
31. With the tubes still in place on the magnetic stand, add 200μL
of 70% ethanol and incubate for 1 min or until solution
becomes clear. Remove ethanol and wash and repeat.
Direct WGS of M. tb’
469
14. Add 20μL of A-Tailing Mix to each 30μL End Repaired Sample
in 8-strip tube, mix by gentle pipetting, and close strip caps.
15. Incubate on a thermocycler at 37
C for 30 min; do not use a
heated lid.
16. Add 90μL of homogenous AMPure XP beads (see Note 9) to
each A-Tailed sample tube (~50μL), mix by pipetting until the
solution becomes homogenous, and incubate at room temperature for 5 min.
17. Place strip tubes on DynaMag-96 Side magnet and allow the
solution to clear (3–5 min).
18. Keeping the tubes on the magnetic stand, carefully remove the
cleared supernatant without disturbing the beads.
19. With the tubes still in place on the magnetic stand, add 200μL
of 70% ethanol and incubate for 1 min or when solution clears.
Remove ethanol and wash and repeat.
20. After second wash step use a P10 tip to remove any residual
ethanol from the bottom of the tubes.
21. Dry samples on thermocycler at 37 ˚C for not more than 5 min
(see Note 10).
22. Add 15μL of nuclease-free water directly to bead pellet. Mix by
vortexing and briefly spin tubes to collect contents. Incubate
for 2 min at room temperature.
23. Put tubes on magnetic rack, leave for 2–3 min to clear, collect
the supernatant (~13μL), and transfer to a new 8-strip tube.
24. Proceed immediately to adaptor ligation step.
25. Prepare Adaptor Ligation Mix for each sample, on ice.
26. Add 37μL of Adaptor Ligation Mix to each 13μL A-Tailed
Sample in an 8-tube strip, mix by gentle pipetting, and close
strip caps.
27. Incubate on a thermocycler at 20 ˚C for 15 min and do not use
a heated lid.
28. Add 90μL of homogenous AMPure XP beads (see Note 9) to
each Adapter Ligated sample tube (~50μL), mix by pipetting
until the solution becomes homogenous, and incubate at room
temperature for 5 min.
29. Place strip tubes on DynaMag-96 Side magnet and allow the
solution to clear (3–5 min).
30. Keeping the tubes on the magnetic stand and carefully remove
the cleared supernatant without disturbing the beads.
31. With the tubes still in place on the magnetic stand, add 200μL
of 70% ethanol and incubate for 1 min or until solution
becomes clear. Remove ethanol and wash and repeat.
Direct WGS of M. tb’
469