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:
- 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°
- 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!
- 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°
- 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
- 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!
- 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
- Add 1 µl DNase (from the Ambion kit) to remove template DNA and incubate at 37° for 15 minutes.
- Stop reaction with 0.8 µl 0.5M EDTA
- Freeze the label probe at -20° (-80° is probably better) if not using right away.
Pre Hybridizing and Hybridizing using RNA probe
- Mix up hybridization solution:
[Stock] Reagent Amount
for 10 ml
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 —
- Prehyb filter in 25 ml prehyp solution for at least 4 hours at 65°
- Add 10 µl of your labelled probe mix per 5ml of Hyb solution.
- Pour off prehyb solution, add hyb solution, and hybridize O/N at 65°
- After hybridization, wash membrane twice in 2X washing solution for 15min/wash at room temp.
2X Wash Solution (200 ml) [Stock] Reagent Amount [Final] 20X SSC 20 ml 2X 5% 4 ml 0.1% — DEPC water 176ml —
- 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 —
- Membrane is now ready for detection.
All steps done at room temperature
- After post hyb washes, equilibrate the membrane in washing buffer for 1 minute.
for 100 ml
for 500 ml
[Final] Tween 20 0.3 ml 1.5 ml 0.3% Maleic Acid 99.7 ml 498.5 —
- 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 —
- Dilute the anti-Digoxigenin AP 1:10,000 (3µl antibody in 30ml Blocking Buffer). Be sure to spin down the antibody before use.
- Pour off block solution and incubate membrane for 30 min in the antibody solution, with shaking, at RT.
- Pour off antibody solution and wash the membrane two times in Washing Buffer, 15min/wash
- 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 —
- Dilute CDP-Star 1:100 in detection buffer (must be at RT)
- Apply substrate to membrane dropwise, and seal in a Seal-a-Meal bag.
- Expose to film.
|50 gm||AG 501-X8 Ion Exchange Resin|
|Stir 30 min, then filter and store at 4°|
Maleic Acid Buffer
for 500 ml
for 1000 ml
|—||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.|
for 100 ml
for 500 ml
|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|
|DEPC water||to 100 ml|
This page is, as always, Copyright (c) 1998 by Craig D. Amundsen. All rights reserved.