9. Put tube on magnetic rack, leave for 2–3 min to clear and
collect the supernatant (~50μL), and transfer to a new DNA
LoBind tube.
10. Add 3μL of D1000 Sample Buffer and 1μL of D1000 ladder to
tube 1 of an 8-tube strip (see Note 11).
11. Add 3μL of D1000 Sample Buffer and 1μL of cleaned, sheared
sample to tubes 2–16 using 8-tube strips (see Note 12), add
caps and vortex, and spin down for 1 min.
12. Load samples into TapeStation, and run D1000 tape. Assess
quality and quantity, with a peak height between 150 and
200 nucleotides.
13. Samples can be stored overnight in the fridge at 4
C or at
À20
C for longer terms.
3.4 End Repair,
A-Tailing, and Adapter
Ligation
1. Prepare End Repair Mix for each sample, on ice.
2. Pipette 52μL of End Repair Mix into thin-wall 8-strip tubes,
add 48μL of each sheared and cleaned DNA sample, mix by
gentle pipetting, and close strip caps.
3. Incubate on a thermocycler at 20 ˚C for 30 min; do not use a
heated lid.
4. Add 180μL of homogenous AMPure XP beads (see Note 9) to
each End Repaired sample tube (~100μL), mix by pipetting
until the solution becomes homogenous, and incubate at room
temperature for 5 min.
5. Place strip tubes on DynaMag-96 Side magnet and allow the
solution to clear (3–5 min).
6. Keeping the tubes on the magnetic stand, carefully remove the
cleared supernatant without disturbing the beads.
7. 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.
8. After second wash step use a P10 tip to remove any residual
ethanol from the bottom of the tube.
9. Dry samples on thermocycler at 37
C for not more than 5 min
(see Note 10).
10. Add 32μL 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.
11. Put tubes on magnetic rack, leave for 2–3 min to clear and
collect the supernatant (~30μL), and transfer to a new
8-strip tube.
12. Samples can be stored overnight in the fridge at 4
C or at
À20
C for longer terms.
468
Amanda Claire Brown
collect the supernatant (~50μL), and transfer to a new DNA
LoBind tube.
10. Add 3μL of D1000 Sample Buffer and 1μL of D1000 ladder to
tube 1 of an 8-tube strip (see Note 11).
11. Add 3μL of D1000 Sample Buffer and 1μL of cleaned, sheared
sample to tubes 2–16 using 8-tube strips (see Note 12), add
caps and vortex, and spin down for 1 min.
12. Load samples into TapeStation, and run D1000 tape. Assess
quality and quantity, with a peak height between 150 and
200 nucleotides.
13. Samples can be stored overnight in the fridge at 4
C or at
À20
C for longer terms.
3.4 End Repair,
A-Tailing, and Adapter
Ligation
1. Prepare End Repair Mix for each sample, on ice.
2. Pipette 52μL of End Repair Mix into thin-wall 8-strip tubes,
add 48μL of each sheared and cleaned DNA sample, mix by
gentle pipetting, and close strip caps.
3. Incubate on a thermocycler at 20 ˚C for 30 min; do not use a
heated lid.
4. Add 180μL of homogenous AMPure XP beads (see Note 9) to
each End Repaired sample tube (~100μL), mix by pipetting
until the solution becomes homogenous, and incubate at room
temperature for 5 min.
5. Place strip tubes on DynaMag-96 Side magnet and allow the
solution to clear (3–5 min).
6. Keeping the tubes on the magnetic stand, carefully remove the
cleared supernatant without disturbing the beads.
7. 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.
8. After second wash step use a P10 tip to remove any residual
ethanol from the bottom of the tube.
9. Dry samples on thermocycler at 37
C for not more than 5 min
(see Note 10).
10. Add 32μL 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.
11. Put tubes on magnetic rack, leave for 2–3 min to clear and
collect the supernatant (~30μL), and transfer to a new
8-strip tube.
12. Samples can be stored overnight in the fridge at 4
C or at
À20
C for longer terms.
468
Amanda Claire Brown