DIG Labeled RNA Probe Protocol

This protocol is used to find ends of BACs to do a chromosome walk

Adapted from a protocol I got from Rachel Nguyen

BAC DNA Digestion and Purification:

  1. Digest DNA with an enzyme that cuts Chlamy DNA often: (e.g. PvuII)

    Incubate O/N at 37°

  2. Add 1 µl 10mg/ml RNase to the reaction mix and incubate for 1 hour at 37°
  3. From this point on, wear gloves and treat everything like you are working with RNA!

  4. After incubation, extract twice with 25 µl of Phenol/CHCl3
  5. To the aqueous layer add 8.9 µl 1M Na2OAc and 67.8 µl EtOH and precipitate O/N at -20°

  6. Spin for 15 minutes at 4° in the microfuge, wash pellet with 70% EtOH (made with DEPC water), and resuspend pellet in 5 µl DEPC treated TE
  7. Let resuspend O/N at 4°. This will be the DNA you will use to make the probe.

Labeling the RNA probe:

From this point on use only DEPC treated solutions or solutions made with DEPC water!

  1. Add reagents (from the Ambion kit):

    Incubate at 37° for 1 hour

  2. Add 1 µl DNase (from the Ambion kit) to remove template DNA and incubate at 37° for 15 minutes.
  3. Stop reaction with 0.8 µl 0.5M EDTA
  4. Freeze the label probe at -20° (-80° is probably better) if not using right away.

Pre Hybridizing and Hybridizing using RNA probe

  1. Mix up hybridization solution:

  2. Prehyb filter in 25 ml prehyp solution for at least 4 hours at 65°
  3. Add 10 µl of your labelled probe mix per 5ml of Hyb solution.
  4. Pour off prehyb solution, add hyb solution, and hybridize O/N at 65°
  5. After hybridization, wash membrane twice in 2X washing solution for 15min/wash at room temp.

  6. Wash membrane twice in 0.5X washing solution for 15min/wash at 60°.

  7. Membrane is now ready for detection.


All steps done at room temperature

  1. After post hyb washes, equilibrate the membrane in washing buffer for 1 minute.

  2. Shake the membrane in Blocking Buffer for 30-60 minutes. (Longer times are okay)

  3. Dilute the anti-Digoxigenin AP 1:10,000 (3µl antibody in 30ml Blocking Buffer). Be sure to spin down the antibody before use.
  4. Pour off block solution and incubate membrane for 30 min in the antibody solution, with shaking, at RT.
  5. Pour off antibody solution and wash the membrane two times in Washing Buffer, 15min/wash
  6. Equilibrate membrane in detection buffer for 2 minutes

  7. Dilute CDP-Star 1:100 in detection buffer (must be at RT)
  8. Apply substrate to membrane dropwise, and seal in a Seal-a-Meal bag.
  9. Expose to film.

Deinonized Formamide

Maleic Acid Buffer


5% SDS

This page is, as always, Copyright (c) 1998 by Craig D. Amundsen. All rights reserved.