Generating DIG-labeled anti-sense probe for in situ hybridization

1. Begin with linearized plasmid DNA (~2.5 µg).

1X Reaction

   5 µl 10X Reaction Buffer

   5 µl 100 mM DTT

 10 µl 2.5 mM NTP Mix*

   3 µl DNA (2.5 µg)

   3 µl Polymerase (T7, T3, etc.)

   0.5 µl RNasin

 23.5 µl H₂O

50 µl total

2. Incubate reaction for 2 hours at 37°C.

3. Check 1 µl of the reaction on an agarose gel (RNA band should be ~10X more abundant than template DNA band).

4. Add 1 µl DNAse; incubate at 37°C for 15 min.

5. Add 25 µl LiCl; incubate at -20°C for 30 min.

6. Microcentrifuge for 15 min at maximum speed; remove supernatant.

7. Re-suspend in 100 µl H₂O; add 15 µl Ammonium Acetate and 100 µl Isopropanol; incubate 30 min at -20°C; microcentrifuge for 15 min at max. speed.

8. Remove supernatant and wash RNA pellet with 1 mL 70% EtOH; re-suspend in 11 µl DEPC-treated H₂O.

*2.5 mM Nucleotide mix with Digoxigenin-11-UTP (Roche # 1209256; 250 nmol, 25 µl)

Add the following to the tube containing 25 µl of Dig-11-UTP:

7.1 µl 100 mM CTP, GTP, ATP

4.6 µl 100 mM UTP

234 µl DEPC H₂O

Notes:

When adding probe to Hybridization start with 1 µg/mL concentration then adjust if necessary.

Digoxigenin-labeled riboprobes are stable for at least 2 years when stored at -20°C.