Digoxigenin-UTP RNA labeling

1. Add the following to a sterile microfuge tube on ice:

-1 ug linearized plasmid DNA.

-2 ul Dig RNA Labeling Mix, 10X concentrate

-2 ul 10X transcription buffer

-1-2 ul RNase inhibitor (DNase free)

-add DEPC treated H2O up to 18 ul

-2 ul RNA polymerase as appropriate

2. Mix then spin down briefly.

3. Incubate at 37'C for 2-3 hours.

4. Add 2 ul DNase I, RNase free to remove DNA template.

5. Incubate at 37'C for 15 minutes.

10X NTP Labeling Mix: 10mM ATP, CTP, GTP, 6.5mM UTP, 3.5mM DIG in Tris-HCl,pH 7.5.

10X Transcription Buffer:

400mM Tris-HCl, pH 8.0; 60mM MgCl2, 100mM dithioerythritol, 20mM spermidine, 100mM NaCl, 1U/ml RNase Inhibitor.

Ethanol Precipitation of RNA transcripts:

Note: Use DEPC treated H2O to make reagents.

1. Add 2.5 ul 3M Sodium Acetate and 75 ul 100% ethanol (-20'C) to the above reaction.

2. Mix well then store at -70'C for at least 30 minutes.

3. Centrifuge at 12,000 X g at 4'C for 15 minutes.

4. Wash the pellet carefully with 70% ETOH DEPC treated and(-20'C).

5. Repeat centrifugation. Aspirate supernatant. Dry under vacuum.

6. Dissolve in 10 ul DEPC treated H2O for acrylamide purification or in 100 ul of 0.5% SDS for direct use in situ.

Note: Riboprobes are best stored for long periods of time in ETOH at -70'C.

Determination of Labeling efficiency:

1. Make serial dilutions the transcription reaction in the range of 100 pg/ul to 0.1 pg/ul in sterile, RNase free water and spot on BM positively charged membrane.

2. UV cross link.

3. See "Detection of DIG labeled nucleic acids".