In this step, Reverse hGW19K primers (Rev GSPs) are hybridized with target mRNAs, and mRNA-Rev GSP hybrids are purified from non-hybridized primers by SPRIselect magnetic beads. The protocol assumes that the reactions will be set in a 96-well PCR plate. For small numbers of samples, reactions can be done in tubes or strips.
- Load the plate in the thermal cycler, and run the following program to hybridize mRNAs with Reverse hGW19K primers:
Temperature Time 70°C 5 min 60°C 60 min 25°C ∞
- Add 24 µl (1.2x volume) of SPRIselect magnetic beads (adjusted to room temperature) to each reaction well, seal the plate, and mix SPRIselect beads and hybridization solution in wells using Eppendorf plate shaker. Importantly, you need to optimize the speed of the plate shaker to completely mix viscous SPRIselect beads with solution in each well. Check visually that each well contains a homogeneous brown color solution in the whole volume.
- Incubate the mixture for 5-10 minutes at room temperature. While waiting, prepare the Reverse Transcriptase Master Mix based on protocol in Step 5.3. Store on ice before use.
- Place the plate in the Magnetic Stand for 96-well plates for 1-2 minutes or until the solution is clear. Carefully remove and discard the supernatant without disturbing the pellet.
- Add 200 μl of freshly prepared 80% ethanol in each reaction well without removal of the plate from the Magnetic Stand, wait for 2 minutes, carefully remove, and discard the supernatant without disturbing the bead pellets.
- Repeat 80% ethanol washing as in the previous Step.
- Briefly centrifuge the plate at low speed and place the plate in the Magnetic Stand. Use a 20-μl pipette to remove the residual ethanol droplets from reaction wells and air-dry the beads at room temperature for approximately 5 minutes. Visually check that no residual ethanol droplets are present in the wells. Avoid over-drying the SPRI beads.
Last modified:
4 September 2024
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