32. After second wash step use a P10 tip to remove any residual
ethanol from the bottom of the tubes.
33. Dry samples on thermocycler at 37 ˚C for not more than 5 min
(see Note 10).
34. Add 47μL of nuclease-free water directly to bead pellet. Mix by
vortexing and briefly spin tube to collect contents. Incubate for
2 min at room temperature.
35. Put tubes on magnetic rack, leave for 2–3 min to clear and
collect the supernatant (~45μL), and transfer to a new 8-tube
strip.
36. Add 3μL of D1000 Sample Buffer and 1μL of D1000 ladder to
tube 1 to a new 8-tube strip (see Note 11).
37. Add 3μL of D1000 Sample Buffer and 1μL of 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.
38. Load samples into TapeStation and run D1000 tape. Assess
quality and quantity; the peak height should have increased to
~250–275 bp.
39. Samples can be stored overnight in the fridge at 4
C or at
À20
C for longer terms.
3.5 Amplification
of Adapter Ligated
Library
1. Adjust adaptor ligated samples to 20 ng/μL using nucleasefree water.
2. Prepare Precapture PCR Mix.
3. Add 35μL of Precapture PCR Mix to each 15μL Adaptor
Ligated Sample in 8-strip tube, mix by gentle pipetting, and
close strip caps.
4. Cycle on a thermocycler using the parameters in Table 1 and
repeat Table 1 steps 2–4 for 6–12 cycles (see Note 13).
5. Add 90μL of homogenous AMPure XP beads (see Note 9) to
each Precapture PCR tube (~50μL), mix by pipetting until the
solution becomes homogenous, and incubate at room temperature for 5 min.
6. Place strip tubes on DynaMag-96 Side magnet and allow the
solution to become clear (3–5 min).
7. Keeping the tubes on the magnetic stand, carefully remove the
cleared supernatant without disturbing the beads.
8. With the tubes still in place on the magnetic stand, add 200μL
of 70% ethanol, incubate for 1 min or when solution becomes
clear. Remove ethanol and wash and repeat.
9. After second wash step use a P10 tip to remove any residual
ethanol from the bottom of the tube.
470
Amanda Claire Brown
ethanol from the bottom of the tubes.
33. Dry samples on thermocycler at 37 ˚C for not more than 5 min
(see Note 10).
34. Add 47μL of nuclease-free water directly to bead pellet. Mix by
vortexing and briefly spin tube to collect contents. Incubate for
2 min at room temperature.
35. Put tubes on magnetic rack, leave for 2–3 min to clear and
collect the supernatant (~45μL), and transfer to a new 8-tube
strip.
36. Add 3μL of D1000 Sample Buffer and 1μL of D1000 ladder to
tube 1 to a new 8-tube strip (see Note 11).
37. Add 3μL of D1000 Sample Buffer and 1μL of 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.
38. Load samples into TapeStation and run D1000 tape. Assess
quality and quantity; the peak height should have increased to
~250–275 bp.
39. Samples can be stored overnight in the fridge at 4
C or at
À20
C for longer terms.
3.5 Amplification
of Adapter Ligated
Library
1. Adjust adaptor ligated samples to 20 ng/μL using nucleasefree water.
2. Prepare Precapture PCR Mix.
3. Add 35μL of Precapture PCR Mix to each 15μL Adaptor
Ligated Sample in 8-strip tube, mix by gentle pipetting, and
close strip caps.
4. Cycle on a thermocycler using the parameters in Table 1 and
repeat Table 1 steps 2–4 for 6–12 cycles (see Note 13).
5. Add 90μL of homogenous AMPure XP beads (see Note 9) to
each Precapture PCR tube (~50μL), mix by pipetting until the
solution becomes homogenous, and incubate at room temperature for 5 min.
6. Place strip tubes on DynaMag-96 Side magnet and allow the
solution to become clear (3–5 min).
7. Keeping the tubes on the magnetic stand, carefully remove the
cleared supernatant without disturbing the beads.
8. With the tubes still in place on the magnetic stand, add 200μL
of 70% ethanol, incubate for 1 min or when solution becomes
clear. Remove ethanol and wash and repeat.
9. After second wash step use a P10 tip to remove any residual
ethanol from the bottom of the tube.
470
Amanda Claire Brown