7. Prepare sufficient Hybridization Buffer for the samples being
hybridized.
8. While maintaining PLATE A at 65
C, load 20μL of Hybridization Buffer into the required wells in column 1.
9. Add strip caps to column 1 and seal well; carefully check
each cap to ensure a complete seal.
10. Close thermocycler lid and incubate at 65 ˚C for at least 5 min.
11. Prepare SureSelect RNA Capture Baits by diluting with 10%
RNase Block, for each sample: 2μL of RNA baits (<3.0 MB),
0.5μL of RNase Block, and 4.5μL of nuclease-free water; keep
on ice.
12. In a new 96-well plate (PLATE B) on ice, add SureSelect RNA
Capture Baits (7μL) to column 3.
13. Using a multichannel pipette, transfer 7μL of SureSelect RNA
Capture Baits from column 3 of PLATE B, to column 3 of
PLATE A, still in place on the thermocycler at 65 ˚C.
14. Add strip caps to column 3 of PLATE A and seal well; carefully
check each cap to ensure a complete seal.
15. Close thermocycler lid and incubate at 65 ˚C for 2 min.
16. While maintaining PLATE A at 65 ˚C, remove caps from
column 1 and column 2 and quickly transfer 13μL of Hybridization Buffer from column 1 to column 3, using a multichannel pipette (see Note 17).
17. While continuing to maintain PLATE A at 65
C, remove caps
from column 3 and quickly transfer the entire contents of
column 2 to column 3, using a multichannel pipette, still set
at 13μL (see Note 17).
18. Mix column 3 well by carefully pipetting 8–10 times.
19. Add new strip caps to column 3 of PLATE A and seal well;
carefully check each cap to ensure a complete seal (see Note
18).
20. Add strip caps to columns 1, 2, and 12 of PLATE A, to ensure
the plate is balanced under the heated lid.
21. Incubate the hybridization mixture on the thermocycler for
16–24 h at 65
C.
22. After incubation prepare 50μL of Dynabeads MyOne Streptavidin T1 beads for each hybridized sample, add to a DNA
LoBind tube, and place on magnetic rack for 1–2 min.
23. Remove the cleared supernatant.
24. Add 200μL of SureSelect Binding Buffer.
25. Vortex for 5 s, spin down to collect tube contents, and return
to magnetic rack for 1–2 min.
472
Amanda Claire Brown
hybridized.
8. While maintaining PLATE A at 65
C, load 20μL of Hybridization Buffer into the required wells in column 1.
9. Add strip caps to column 1 and seal well; carefully check
each cap to ensure a complete seal.
10. Close thermocycler lid and incubate at 65 ˚C for at least 5 min.
11. Prepare SureSelect RNA Capture Baits by diluting with 10%
RNase Block, for each sample: 2μL of RNA baits (<3.0 MB),
0.5μL of RNase Block, and 4.5μL of nuclease-free water; keep
on ice.
12. In a new 96-well plate (PLATE B) on ice, add SureSelect RNA
Capture Baits (7μL) to column 3.
13. Using a multichannel pipette, transfer 7μL of SureSelect RNA
Capture Baits from column 3 of PLATE B, to column 3 of
PLATE A, still in place on the thermocycler at 65 ˚C.
14. Add strip caps to column 3 of PLATE A and seal well; carefully
check each cap to ensure a complete seal.
15. Close thermocycler lid and incubate at 65 ˚C for 2 min.
16. While maintaining PLATE A at 65 ˚C, remove caps from
column 1 and column 2 and quickly transfer 13μL of Hybridization Buffer from column 1 to column 3, using a multichannel pipette (see Note 17).
17. While continuing to maintain PLATE A at 65
C, remove caps
from column 3 and quickly transfer the entire contents of
column 2 to column 3, using a multichannel pipette, still set
at 13μL (see Note 17).
18. Mix column 3 well by carefully pipetting 8–10 times.
19. Add new strip caps to column 3 of PLATE A and seal well;
carefully check each cap to ensure a complete seal (see Note
18).
20. Add strip caps to columns 1, 2, and 12 of PLATE A, to ensure
the plate is balanced under the heated lid.
21. Incubate the hybridization mixture on the thermocycler for
16–24 h at 65
C.
22. After incubation prepare 50μL of Dynabeads MyOne Streptavidin T1 beads for each hybridized sample, add to a DNA
LoBind tube, and place on magnetic rack for 1–2 min.
23. Remove the cleared supernatant.
24. Add 200μL of SureSelect Binding Buffer.
25. Vortex for 5 s, spin down to collect tube contents, and return
to magnetic rack for 1–2 min.
472
Amanda Claire Brown