DIG Labeled RNA Probe Protocol

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!

3. 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°

4. 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
5. 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