3' end labeling is useful for analyzing RNA structure with nucleases, as well as for mapping protein binding sites. Below is a protocol for 3' end labeling RNA using T4 RNA ligase.
- T4 RNA Ligase (Ambion Cat #2140)
- 10X T4 RNA Ligase Buffer (supplied with Ambion's T4 RNA Ligase: 0.5 M Tris-HCl, pH 7.8, 0.1 M MgCl2, 0.1 M DTT, 10 mM ATP)
- RNase-free Sephadex G-25 or G-50 spin columns such as Ambion's NucAway™ Spin Columns (Cat #10070)
Combine the following in a single RNase-free microfuge tube:
- 2 µl 10X T4 RNA Ligase Buffer
- 50-100 pmol RNA
- equimolar amount (50-100 pmol) [32P]pCp
- RNase-free water to a final volume of 18 µl
- Add 2 µl T4 RNA Ligase (10 U).
- Incubate at 4¬ C overnight (10-12 hours).
- Remove unincorporated label by applying the mixture to an RNase-free Sephadex G-25 or G-50 spin column (e.g., NucAway Spin Columns) following the manufacturer's recommendations.
Typically, ~10% of the radiolabel will be incorporated in the 3' end-label reaction.