Feb 05, 2019
Preparation of the adenylated oligonucleotide for mapping 3′OH RNA termini
- 1Université de Montréal, Montreal, Quebec, Canada
Protocol Citation: Matus Valach 2019. Preparation of the adenylated oligonucleotide for mapping 3′OH RNA termini. protocols.io https://dx.doi.org/10.17504/protocols.io.xspfndn
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: February 05, 2019
Last Modified: February 05, 2019
Protocol Integer ID: 20015
Keywords: RNA, 3′ RACE, adenylation, RNA-Seq
Abstract
Adapted from the protocol for oligonucleotide 5′ adenylation (E2610) of New England Biolabs (NEB).
Materials
MATERIALS
5’ DNA Adenylation Kit - 10 rxnsNew England BiolabsCatalog #E2610S
Mix the following components (20 μL):
| Component | Amount [μL] | Final concentration | |
| 5′ phosphorylated DNA oligo [50 μM] | 4 | 10 μM | |
| 10× 5′ DNA Adenylation buffer | 2 | 1× | |
| ATP [1 mM] | 2 | 0.1 mM | |
| Mth RNA ligase [50 pmol/µL] | 4 | 10 µM | |
| RNase-free water (ddH2O) | 8 |
Incubate for 60 minutes at 65 °C.
Incubate for 5 minutes at 85 °C to inactivate the enzyme.
The adenylated oligo can be directly used for RNA ligation with T4 RNA ligase 2 (optimally with T4 RNA Ligase 2, truncated KQ (NEB #M0373)).