- Aliquot 20μL of the PCR reaction mix into eight separate
0.2 mL nuclease-free PCR tubes. - Amplify via the following protocol (seeNote 18):
(a) 94C: 2 min.
(b) 94C: 10 s.
(c) 40C: 2 min.
(d) 72C: 1 min.
(e) Go tostep bonce.
(f) 94C: 10 s.
(g) 55C: 30 s.
(h) 72C: 1 min.
(i) Go tostep fseven times.
(j) 94C: 15 s.
(k) 55C: 30 s.
(l) 72C: 1 min.
(m) Go tostep jfourteen times.
(n) 72C: 5 min.
(o) 4C: pause. - Warm Agencourt RNAClean XP beads and PCR samples to
room temperature before proceeding (15 min). - Combine individual PCRs for each sample into a single
1.75 mL microcentrifuge tube (seeNote 19). - Measure the volume of each sample using a pipette.
- Mix the Agencourt RNAClean XP beads well by vortexing.
- Add 0.8 volumes (relative to sample volume) of the pre-
warmed, mixed beads to each sample (seeNote 20). - Mix thoroughly by pipetting>10 times. Vortex gently.
- Incubate at room temperature for 15 min. During the incuba-
tion prepare a fresh 80% ethanol solution. - Place the tube on a magnetic stand for>5 min.
- Remove the supernatant without disturbing the beads.
- With the tube still on the stand, add 400μL of fresh 80%
ethanol without disturbing the beads. - Incubate at room temperature for 1 min while still on the
magnetic stand. - Remove the ethanol supernatant.
- Repeat the 80% ethanol wash for a total of two wash steps.
- Allow the tube to air dry on the magnetic stand (seeNote 13).
- Add 100μL of nuclease-free water to the tube and immediately
and thoroughly mix.
RNA Tagging Library Preparation 467