• My NEB
  • Print
  • PDF
  • Protocol for mRNA (E7350)

    Introduction

    Starting Material: 50–250 ng of mRNA fragmented to 150–250 bp.

    First Strand Synthesis, Second Strand Synthesis, End Repair and dA-Tailing steps are performed following NEBNext Sample Prep Reagent Set 1 (NEB #E6100) or Master Mix Set 1 (NEB #E6110) protocols.

    Protocol

    1. Adaptor Ligation of dA-Tailed DNA
      1. Mix the following components in a sterile microfuge tube:
        Reagent Set Protocol (NEB #E6100)
        Purified, dA-Tailed DNA   23 μl
        Quick Ligation Reaction Buffer (2X)   25 μl
        Adaptor   1 μl
        Quick T4 DNA Ligase   1 μl
        --------------------------------------------------------------
        Total volume should be   50 μ

        Master Mix Protocol (NEB #E6110)
        Purified, dA-Tailed DNA   38 μl
        Quick Ligation Reaction Buffer (5X)   10 μl
        Adaptor   1 μl
        Quick T4 DNA Ligase   1 μl
        --------------------------------------------------------------
        Total volume should be   50 μl
      2. Incubate in a thermal cycler for 15 minutes at 20°C.
      3. Add 3 μl of USER Enzyme, mix by pipetting up and down, and incubate at 37°C for 15 minutes.
      4. Vortex AMPure XP beads to resuspend.
      5. Add 90 μl of resuspended AMPure XP beads the ligation reaction (~ 53 μl). Mix well on a vortex mixer or by pipetting up and down at least 10 times.
      6. Incubate for 5 minutes at room temperature.
      7. Place the tube on an appropriate magnetic stand to separate beads from supernatant. After the solution is clear (about 5 minutes), carefully remove and discard the supernatant. Be careful not to disturb the beads that contain DNA targets.
      8. Add 200 μl of fresh 80% ethanol to the tube while in the magnetic stand. Mix by pipetting up and down. Incubate at room temperature for 30 seconds, and then carefully remove and discard the supernatant.
      9. Repeat Step 8 once.
      10. Air dry the beads for 10 minutes while the tube is on the magnetic stand with lid open.
      11. Elute the DNA target from the beads into 150 μl water for bead-based size selection as detailed in the next section, or at desired volume for size selection using E-Gel size select gels or standard 2% agarose gels. Mix well on a vortex mixer or by pipetting up and down, and put the tube in the magnetic stand until the solution is clear.
      12. Remove 150 μl supernatant (or desired volume for alternate size selection) to a fresh 1.5 ml LoBind tube and proceed to bead based size selection.
    2. Alternately, adaptor ligated DNA can be purified on one purification column. Elute in sterile H2O in volume desired for subsequent size selection.

      Size Select Adaptor Ligated DNA Using AMPure XP Beads

      Caution: The following size selection protocol is for libraries with 200 bp inserts only. For libraries with larger fragment inserts, please optimize bead: DNA ratio accordingly.
      1. Add 135 μl (0.9X) resuspended AMPure XP beads to 150 μl DNA solution. Mix well on a vortex mixer or by pipetting up and down at least 10 times.
      2. Incubate for 5 minutes at room temperature.
      3. Place the tube on an appropriate magnetic stand to separate beads from supernatant. After the solution is clear (about 5 minutes), carefully transfer the supernatant to a new tube (Caution: do not discard the supernatant). Discard beads that contain the large fragments.
      4. Add 30 μl (0.2X) resuspended AMPure XP beads to the supernatant, mix well and incubate for 5 minutes at room temperature.
      5. Place the tube on an appropriate magnetic stand to separate beads from supernatant. After the solution is clear (about 5 minutes), carefully remove and discard the supernatant. Be careful not to disturb the beads that contain DNA targets (Caution: do not discard beads).
      6. Add 200 μl of fresh 80% ethanol to the tube while in the magnetic stand. Incubate at room temperature for 30 seconds, and then carefully remove and discard the supernatant.
      7. Repeat Step 6 once.
      8. Briefly spin the tube, and put the tube back in the magnetic stand. Completely remove the residual ethanol, and air dry beads for 10 minutes while the tube is on the magnetic stand with lid open.
      9. Elute DNA target from beads into 22 μl nuclease free water or 0.1X TE buffer. Mix well on a vortex mixer or by pipetting up and down, and put the tube in the magnetic stand until the solution is clear.
      10. Transfer 20 μl of the supernatant to a clean PCR tube, and proceed to enrichment. 

        Alternatively, adaptor ligated DNA can be size selected using a number of other methods including E-Gel size select gels or standard 2% agarose gels. Purify DNA sample on one column and elute in 22 μl of sterile water or elution buffer.
    3. PCR Enrichment of Adaptor Ligated DNA
      1. Mix the following components in a sterile microfuge tube: 

        DNA   20 μl
        Universal PCR Primer (25 μM)   1 μl
        Index Primer 1 (25 μM)   1 μl
        Phusion High Fidelity Reaction Buffer (5X)   10 μl
        dNTP Solution Mix   1.5 μl
        Phusion High Fidelity DNA Polymerase   0.5 μl
        Nuclease-free Water   16 μl 
        ---------------------------------------------------------------------
        Total volume   50 μl
      2. PCR cycling conditions:
        Cycle Step Temp Time Cycles
        Initial Denatuation 98°C 10 seconds 1
        Denaturation
        Annealing
        Extension
        98°C
        65°C
        72°C
        10 seconds
        30 seconds
        30 seconds
        10–12*
        Final Extension 72°C 5 minutes 1
        Hold 4°C  
        *The number of PCR cycles should be adjusted based on mRNA input. If 50 ng of purified mRNA is the starting input, it is recommended to perform 12 cycles of PCR.
      3. Vortex AMPure XP beads to resuspend.
      4. Add 60 μl (1.2X) of resuspended AMPure XP beads PCR reaction (~ 50 μl). Mix well on a vortex mixer or by pipetting up and down at least 10 times.
      5. Incubate for 5 minutes at room temperature.
      6. Place the tube on an appropriate magnetic stand to separate beads from supernatant. After the solution is clear (about 5 minutes), carefully remove and discard the supernatant. Be careful not to disturb the beads that contain DNA targets.
      7. Add 200 μl of fresh 80% ethanol to the tube while in the magnetic stand. Incubate at room temperature for 30 seconds, and then carefully remove and discard the supernatant.
      8. Repeat Step 7 once
      9. Air dry beads for 10 minutes while the tube is on the magnetic stand with the lid open.
      10. Elute DNA target from beads into 22.5 μl of 0.1X TE. Mix well on a vortex mixer or by pipetting up and down, and put the tube in the magnetic stand until the solution is clear.
      11. Transfer 20 μl of the supernatant to a clean 1.5 ml LoBind tube, and store at -20°C.
        Alternately, adaptor ligated DNA can be purified on one purification column. Elute in 22.5 µl of 0.1X TE Buffer.
      12. Assess library quality on a Bioanalyzer (Agilent high sensitivity chip). Check that the electropherogram shows a narrow distribution with a peak size approximately 270 bp.