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Introduction
T4 RNA ligase catalyzes the ligation of the 5' phosphate terminus of a nucleic acid donor to the 3' OH terminus of a nucleic acid acceptor. Substrates include RNA, DNA, and oligonucleotides. The reaction is ATP dependent. T4 RNA ligase can be used to 3'-end label RNA molecules, as well as to circularize RNA and DNA molecules. The enzyme is used in Ambion's FirstChoice™ RLM-RACE Kit for tagging the 5' ends of mRNA with oligonucleotide adaptor.
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.
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.
Materials
Reagents and Equipment Required
- 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)
- [32P]pCp
- RNase-free Sephadex G-25 or G-50 spin columns such as Ambion's NucAway™ Spin Columns (Cat #10070)
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Protocol for 3' End Labeling RNA
- 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.
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