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  3. RNA 3'-end Labeling by Ligation

Thermo Fisher Scientific RNA 3'-end Labeling by Ligation User guide

  • Hello! I have reviewed the provided user guide for RNA 3'-end labeling by ligation using T4 RNA Ligase. This document details the necessary steps, from preparing the mix in a RNase-free tube to separating the labeled RNA using gel filtration with Sephadex G-50. It includes specifics on reagents, incubation time, and temperature. I'm ready to answer your questions.
  • What is the incubation temperature for the reaction?
    How is labeled RNA separated from unincorporated label?
    What is the required incubation time?
Protocol
1. Prepare the following in a single RNase-free microfuge tube:
10X ligation buffer for T4 RNA Ligase 2 µl
10 mM ATP 1 µl
RNA 50-100 pmol
[32P]-pCp 50-100 pmol (equimolar amount)
T4 RNA Ligase 1 µl (10 u)
DEPC-treated Water to 20 µl
Total volume 20 µl
2. Incubate at 4°C for 10-12 hours (overnight).
3. Separate labeled RNA from unincorporated label by gel ltration on Sephadex G-50.
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RNA 3'-end Labeling by Ligation
This protocol is for the RNA 3'-end Labeling by Ligation
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