- Transcriptome Analysis: Library Construction 21
of the PCR amplified library. 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.
Note: Leave the sample tubes on the magnetic stand whiling 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 32.5μL 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 30μL of the clear supernatant to a new PCR tube.
Note: The purified DNA library can be stored at−15 to− 25 ◦C for up to seven days.Figure 3. Example of TruSeq RNA Sample Prep v2 Library Size Distribution. Electro-
pherograms were obtained with the Agilent 2100 Bioanalyzer (Control, control sample;
Treat, treated sample) before (a) and after (b) library amplification.