DIG-Labeled RNA Probes

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)
      5 µl BAC DNA
      1 µl PvuII (10 units)
      2.5 µl 10X PvuII buffer
      16.5 µl water
      25 µl total

      Incubate O/N at 37°

    2. Add 1 µl 10mg/ml RNase to the reaction mix and incubate for 1 hour at 37°

From this point on, wear gloves and treat everything like you are working with RNA!

    1. After incubation, extract twice with 25 µl of Phenol/CHCl3

To the aqueous layer add 8.9 µl 1M Na2OAc and 67.8 µl EtOH and precipitate O/N at -20°

  1. 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
  2. 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):
    8.5 µl RNase-free water
    2 µl 10X txn buffer
    1 µl 10mM ATP
    1 µl 10mM GTP
    1 µl 10mM CTP
    2.5 µl BAC DNA (from step 1)
    1.5 µl 10mM DIG-11-dUTPj
    2 µl T7 (or SP6) RNA polymerase (20 units)

    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:
    [Stock] Reagent Amount
    for 10 ml    		 Amount
    for 25 ml    		 [Final]
    100% 5 ml 12.5 ml 50%
    20X SSPE (filter sterilized) 2.5 ml 6.25 ml 5X
    50X Denhardt’s (filter sterilized) 2 ml 5 ml 10X
    — SDS 0.4 gm 1.0 gm 4%
    10 mg/ml Salmon Sperm DNA 0.3 ml 0.75 ml 0.3 mg/ml
    — DEPC water to 10 ml to 25 ml —
  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.
        “/bac_probe.html#SDS”>SDS
    2X Wash Solution (200 ml)
    [Stock] Reagent Amount [Final]
    20X SSC 20 ml 2X
    5% 4 ml 0.1%
    — DEPC water 176ml —
  6. Wash membrane twice in 0.5X washing solution for 15min/wash at 60°.
    0.5X Wash (200ml)
    [Stock] Reagent Amount [Final]
    20X SSC 5 ml 0.5X
    5% SDS 4 ml 0.1%
    — DEPC water 191 ml —
  7. Membrane is now ready for detection.

Detection:

All steps done at room temperature

  1. After post hyb washes, equilibrate the membrane in washing buffer for 1 minute.
    Reagent Amount
    for 100 ml    		 Amount
    for 500 ml    		 [Final]
    Tween 20 0.3 ml 1.5 ml 0.3%
    Maleic Acid 99.7 ml 498.5 —
  2. Shake the membrane in Blocking Buffer for 30-60 minutes. (Longer times are okay)
    Reagent Amount [Final]
    Nonfat Dry Milk 2.5gm 5%
    Tween 20 150ul 0.3%
    Maleic Acid 50ml —
  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
    [Stock] Reagent Amount [Final]
    1 M Tris-HCl (pH 9.5) 20ml 100mM
    3 M NaCl 6.67 ml 100 mM
    — DEPC water to 200 ml —
  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

Amount Reagent
50 gm AG 501-X8 Ion Exchange Resin
500 ml formamide
Stir 30 min, then filter and store at 4°

Maleic Acid Buffer

[Stock] Reagent Amount
for 500 ml		 Amount
for 1000 ml		 [Final]
— maleic acid 5.805 gm 11.61 gm 0.1 M
5 M NaCl 15 ml 30 ml 0.15 M
pH to 7.5 water up to 500ml
Treat with DEPC and autoclave.

20X SSC

Reagent Amount
for 100 ml		 Amount
for 500 ml		 [Final]
NaCl 17.53 gm 87.65gm 3 M
sodium citrate 8.82 gm 44.1 gm 300 mM
pH7.0 DEPC water to 100 ml or 500 ml

5% SDS

Reagent Amount
SDS 5 gm
DEPC water to 100 ml

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