Protocol for 5' End Labeling RNA
互联网
实验步骤
1. Protocol for Removing 5' Phosphate
1) Combine the following in a single RNase-free microfuge tube:
-- µl Nuclease-free Water (to make a final volume of 10 µl)
1 µl 10X Dephosphorylation Buffer (0.5 M Tris, pH 8.5, 1 mM EDTA, pH 8)
1 µl Calf Intestinal Phosphatase (CIP; 0.1 U/µl)
3) To the above reaction, add:
15 µl 5.0 M ammonium acetate or 3.0 M sodium acetate
2. Protocol for 5' End Labeling RNA
1) Combine the following in a single RNase-free microfuge tube:
-- µl nuclease-free water (to make a final volume of 20 µl)
-- µl RNA (0.1 to 100 pmol RNA)
25 pmol [gamma-32P]ATP (7000 Ci/mmol, 150 mCi/ml)
2 µl 10X Kinase Buffer (500 mM Tris, pH 7.5, 100 mM MgCl2, 50 mM DTT)
1 µl T4 Polynucleotide Kinase (10 U/ml)
3) (optional) Stop the reaction by adding EDTA to 1 mM, and then heating to 95¬C for 2 minutes.
