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".