26. Repeat steps 23–25 for a total of three washes.
27. Resuspend beads in 200μL of SureSelect Binding Buffer &
label each LoBind tube with sample identifier.
28. Open the lid to the thermocycler containing PLATE A,
keeping the plate at 65
C, open the first cap for column
3 (A3), remove the contents (typically 27–29μL, see Note
19), and place in corresponding LoBind tube containing resuspended MyOne Streptavidin T1 beads. Invert 3–5 times to
mix. Repeat for other samples (B3, C3. . .).
29. Mix all tubes containing hybridized samples and MyOne Streptavidin T1 beads well on a vortex for 5 s and then transfer to a
Nutator. Incubate on the Nutator for 30 min at room temperature (see Note 20).
30. Briefly spin the tubes in a microfuge to collect the contents,
then add to a magnetic rack, and allow to clear for 1–3 min.
31. Remove the cleared supernatant and discard.
32. Add 500μL of SureSelect Wash Buffer #1 to each tube, close
caps, and vortex for 5 s.
33. Incubate at room temperature for 15 min and mix every 5 min
using a vortex.
34. Briefly spin the tubes in a microfuge to collect the contents,
then add to a magnetic rack, and allow to clear for 1–3 min.
35. Remove the cleared supernatant and discard.
36. Add 500μL of prewarmed 65
C SureSelect Wash Buffer #2 (see
Note 21) to each tube, close caps, and vortex for 5 s.
37. Incubate on a heat block at 65
C for 10 min and mix every
3 min using a vortex.
38. Repeat steps 34–37 for a total of 3 SureSelect Wash Buffer #2
washes.
39. After the final wash, briefly spin tube before returning to the
magnetic rack and use a P10 tip to ensure all residual wash
buffer is removed.
40. Resuspend bead pellet in 45μL of nuclease-free water and leave
beads remaining in tube.
41. Samples should be stored at 4
C.
3.7 Addition of Index
Tags by
Post-hybridization
Amplification
1. Prepare Postcapture PCR Mix.
2. Add 34μL of Postcapture PCR Mix to required number of
tubes in 8-strip tube.
3. Resuspend beads in hybridized samples and pipette 15μL of
bead mix into tube containing Postcapture PCR Mix.
4. Add 1μL of the appropriate PCR Index Primer to each reaction
tube (see Note 22), mix by gentle pipetting, and close
strip caps.
Direct WGS of M. tb’
473
27. Resuspend beads in 200μL of SureSelect Binding Buffer &
label each LoBind tube with sample identifier.
28. Open the lid to the thermocycler containing PLATE A,
keeping the plate at 65
C, open the first cap for column
3 (A3), remove the contents (typically 27–29μL, see Note
19), and place in corresponding LoBind tube containing resuspended MyOne Streptavidin T1 beads. Invert 3–5 times to
mix. Repeat for other samples (B3, C3. . .).
29. Mix all tubes containing hybridized samples and MyOne Streptavidin T1 beads well on a vortex for 5 s and then transfer to a
Nutator. Incubate on the Nutator for 30 min at room temperature (see Note 20).
30. Briefly spin the tubes in a microfuge to collect the contents,
then add to a magnetic rack, and allow to clear for 1–3 min.
31. Remove the cleared supernatant and discard.
32. Add 500μL of SureSelect Wash Buffer #1 to each tube, close
caps, and vortex for 5 s.
33. Incubate at room temperature for 15 min and mix every 5 min
using a vortex.
34. Briefly spin the tubes in a microfuge to collect the contents,
then add to a magnetic rack, and allow to clear for 1–3 min.
35. Remove the cleared supernatant and discard.
36. Add 500μL of prewarmed 65
C SureSelect Wash Buffer #2 (see
Note 21) to each tube, close caps, and vortex for 5 s.
37. Incubate on a heat block at 65
C for 10 min and mix every
3 min using a vortex.
38. Repeat steps 34–37 for a total of 3 SureSelect Wash Buffer #2
washes.
39. After the final wash, briefly spin tube before returning to the
magnetic rack and use a P10 tip to ensure all residual wash
buffer is removed.
40. Resuspend bead pellet in 45μL of nuclease-free water and leave
beads remaining in tube.
41. Samples should be stored at 4
C.
3.7 Addition of Index
Tags by
Post-hybridization
Amplification
1. Prepare Postcapture PCR Mix.
2. Add 34μL of Postcapture PCR Mix to required number of
tubes in 8-strip tube.
3. Resuspend beads in hybridized samples and pipette 15μL of
bead mix into tube containing Postcapture PCR Mix.
4. Add 1μL of the appropriate PCR Index Primer to each reaction
tube (see Note 22), mix by gentle pipetting, and close
strip caps.
Direct WGS of M. tb’
473