Size Select the Amplified cDNA Library (E7300)
Protocol
- Mix the purified PCR product (25 μl) with 5 μl of Gel Loading Dye, Blue (6X).
- Load 5 μl of Quick-Load pBR322 DNA-MspI Digest in one well on the 6% PAGE 10-well gel.
- Load two wells with 15 μl each of mixed amplified cDNA construct and loading dye on the 6% PAGE 10-well gel.
- 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.
- 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.
- 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.
- 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).
- Rotate end-to-end for at least 2 hours at room temperature.
- Transfer the eluate and the gel debris to the top of a gel filtration column.
- Centrifuge the filter for 2 min at > 13,200 rpm.
- Recover eluate and add 1 μl Linear Acrylamide, 25 μl 3M sodium acetate, pH 5.5 and 750 μl of 100% ethanol.
- Vortex well.
- Precipitate in a dry ice/methanol bath or at -80°C for at least 30 minutes.
- Spin in a microcentrifuge @ > 14,000 x g for 30 minutes at 4°C.
- Remove the supernatant taking care not to disturb the pellet.
- Wash the pellet with 80% ethanol by vortexing vigorously.
- Spin in a microcentrifuge @ > 14,000 x g for 30 minutes at 4°C.
- Air dry pellet for up to 10 minutes at room temperature to remove residual ethanol.
- Resuspend pellet in 12 μl TE Buffer.
