- Transcriptome Analysis: Library Construction 17
- 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 supernatant (ds cDNA) to a new PCR tube.
Note: The purified ds DNA can be stored at−15 to− 25 ◦C for up to seven days.V.Repair ends
- Pre-heat the thermal cycler to 30◦C.
- Prepare the in-line control reagent by diluting the CTE to 1/100 in RSB
(1μL CTE + 99μL RSB) before use. Add 10μL of dilute CTE to each
sample that contains 50μL of dsDNA. If not using the in-line control
reagent, add 10μL of RSB instead. - Add 40μL of ERP to each sample. Gently pipette the entire volume up
and down 10 times to mix thoroughly. - Place the sample on the thermal cycler to pre-form end repair.
30 ◦C for 30 minutes- Vortex the AMPure XP beads until they are well dispersed, then add
160 μL of well-mixed AMPure XP beads to each sample containing 100μL
of end repaired dsDNA mixture. 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 127.5μL 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.