Digoxigenin-UTP RNA labeling
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".
