- Transcriptome Analysis: Library Construction 19
- Place the sample on the thermal cycler to preform adapter ligation.
30 ◦C for 10 minutes
- Remove samples from thermal cycler. Add 5μL of STL to each sample to
inactivate the ligation. Gently pipette the entire volume up and down 10
times to mix thoroughly.
- Vortex the AMPure XP beads until they are well dispersed, then add
42 μL of well-mixed AMPure XP beads to each sample containing 42.5μL
of sample. Gently pipette the entire volume up and down 10 times to mix
thoroughly.
- Incubate the sample at room temperature for 15 minutes.
- Centrifuge briefly and place the sample on magnetic stand at room
temperature for 5 minutes to make sure that all beads are bound to the
side of the wells.
- Remove and discard 79.5μL of supernatant from each sample.
Note: Leave the sample tubes on the magnetic stand while performing all the following
80% EtOH wash steps.
- With the sample remaining on the magnetic stand, add 200μLoffreshly
prepared 80% EtOH to each sample without disturbing the beads.
- Incubate the sample at room temperature for 30 seconds, then remove
and discard all of the supernatant from each well.
- Repeat the 80% EtOH wash steps twice.
- Open the lid and let the sample tube stand at room temperature for
15 minutes to dry and then remove the sample from the magnetic
stand.
- Add 52.5μL RSB to each sample. Gently pipette the entire volume up
and down 10 times to mix thoroughly.
- Incubate the sample at room temperature for 2 minutes.
- Centrifuge briefly and place the sample on the magnetic stand at room
temperature for 5 minutes.
- Transfer 50μL of the clear supernatant to a new PCR tube.
- Vortex the AMPure XP beads until they are well dispersed, then add
50 μL of well-mixed AMPure XP beads to each sample containing 50μL
of sample. Gently pipette the entire volume up and down 10 times to mix
thoroughly.
- Incubate the sample at room temperature for 15 minutes.
- Centrifuge briefly and place the sample on magnetic stand at room
temperature for 5 minutes to make sure that all beads are bound to the
side of the wells.
- Remove and discard 95μL of supernatant from each sample.
