RNA Detection

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  1. Aliquot 20μL of the PCR reaction mix into eight separate
    0.2 mL nuclease-free PCR tubes.

  2. 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.

  3. Warm Agencourt RNAClean XP beads and PCR samples to
    room temperature before proceeding (15 min).

  4. Combine individual PCRs for each sample into a single
    1.75 mL microcentrifuge tube (seeNote 19).

  5. Measure the volume of each sample using a pipette.

  6. Mix the Agencourt RNAClean XP beads well by vortexing.

  7. Add 0.8 volumes (relative to sample volume) of the pre-
    warmed, mixed beads to each sample (seeNote 20).

  8. Mix thoroughly by pipetting>10 times. Vortex gently.

  9. Incubate at room temperature for 15 min. During the incuba-
    tion prepare a fresh 80% ethanol solution.

  10. Place the tube on a magnetic stand for>5 min.

  11. Remove the supernatant without disturbing the beads.

  12. With the tube still on the stand, add 400μL of fresh 80%
    ethanol without disturbing the beads.

  13. Incubate at room temperature for 1 min while still on the
    magnetic stand.

  14. Remove the ethanol supernatant.

  15. Repeat the 80% ethanol wash for a total of two wash steps.

  16. Allow the tube to air dry on the magnetic stand (seeNote 13).

  17. Add 100μL of nuclease-free water to the tube and immediately
    and thoroughly mix.


RNA Tagging Library Preparation 467
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