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  • mRNA Fragmentation Protocol (E6114)

    Introduction

    Starting Material: mRNA purified from 1–10 μg total RNA

    Protocol

    1. mRNA Fragmentation Protocol
      1. Mix the following components in a sterile PCR tube:
        Purified mRNA: 1–18 μl 
        10X RNA Fragmentation Reaction Buffer:  2 μl 
        Nuclease-Free Water: variable 
        ------------------------------------------------------
        Total volume: 20 μl
      2. Incubate in a preheated thermal cycler for exactly 5 minutes at 94°C.
      3. Transfer tube to ice.
      4. Add 2 μl 10X RNA Fragmentation Stop Solution.
    2. Ethanol Precipitation of Fragmented mRNA
      1. Mix the following components in a sterile 1.5 ml microcentrifuge tube: 
        Fragmented mRNA: 22 μl 
        3 M Sodium Acetate, pH 5.2:  2 μl 
        Linear Acrylamide, 10 mg/ml: 1–2 μl 
        100% Ethanol: 60 μl 
        ------------------------------------------------------
        Total volume: 85–86 μl
      2. Incubate at -80°C for 30 minutes.
      3. Centrifuge at 14,000 rpm for 25 minutes at 4°C in a microcentrifuge.
      4. Carefully remove ethanol.
      5. Wash pellet with 300 μl of 70% ethanol.
      6. Centrifuge and carefully remove 70% ethanol.
      7. Air dry pellet for up to 10 minutes at room temperature to remove residual ethanol.
      8. Resuspend in 13.5 μl Nuclease-Free Water.
    3. Alternative Protocol:
      Clean up Fragmented RNA using a RNA column purification kit such as Qiagen’s RNeasy Minelute Kit, eluting the RNA in 13.5 μl Nuclease-Free Water or elution buffer.