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  • Size Select the Amplified cDNA Library (E7300)

    Protocol

    1. Mix the purified PCR product (25 μl) with 5 μl of Gel Loading Dye, Blue (6X).

    2. Load 5 μl of Quick-Load pBR322 DNA-MspI Digest in one well on the 6% PAGE 10-well gel.

    3. Load two wells with 15 μl each of mixed amplified cDNA construct and loading dye on the 6% PAGE 10-well gel.

    4. Run the gel for 1 hour at 120 V or until the blue dye reaches the bottom of the gel. Do not let the blue dye exit the gel.

    5. Remove the gel from the apparatus and stain the gel with SYBR Gold nucleic acid gel stain in a clean container for 2–3 minutes and view the gel on a UV transiluminator.

    6. The 140 and 150 nucleotide bands correspond to adapter-ligated constructs derived from the 21 and 30 nucleotide RNA fragments, respectively. For miRNAs, isolate the bands corresponding to ~140 bp. For piRNAs, isolate the band corresponding to ~150 bp.

    7. Place the two gel slices from the same sample in one 1.5 ml tube and crush the gel slices with the RNase-free Disposable Pellet Pestles and then soak in 250 μl DNA Gel Elution buffer (1X).

    8. Rotate end-to-end for at least 2 hours at room temperature.

    9. Transfer the eluate and the gel debris to the top of a gel filtration column.

    10. Centrifuge the filter for 2 min at > 13,200 rpm.

    11. Recover eluate and add 1 μl Linear Acrylamide, 25 μl 3M sodium acetate, pH 5.5 and 750 μl of 100% ethanol.
    12. Vortex well.

    13. Precipitate in a dry ice/methanol bath or at -80°C for at least 30 minutes.

    14. Spin in a microcentrifuge @ > 14,000 x g for 30 minutes at 4°C.

    15. Remove the supernatant taking care not to disturb the pellet.

    16. Wash the pellet with 80% ethanol by vortexing vigorously.

    17. Spin in a microcentrifuge @ > 14,000 x g for 30 minutes at 4°C.

    18. Air dry pellet for up to 10 minutes at room temperature to remove residual ethanol.

    19. Resuspend pellet in 12 μl TE Buffer.