This step is needed to remove the excess of nucleotides and GSP1 from the first strand product. In presence of a chaotropic agent (sodium iodide), cDNAs with a minimal length of 200 bases are bound to the silica-based membrane. Buffer components, dNTPs, enzymes, and oligonucleotides remain in solution and are removed by centrifugation. Residual impurities and sodium iodide are removed by passing several volumes of 1X wash buffer followed by a 70% through the GlassMAX cartridge. Purified cDNA is recovered in deionized water.
1. Equilibrate the binding solution to room temperature.
2. For each sample to be purified, equilibrate 100 pL of sterilized, deionized water at 65°C for use in step 9.
3. Add 120 pL of binding solution (6 M NaI) to the first strand reaction (see Note 4).
4. Transfer the cDNA/NaI solution to a GlassMAX spin cartridge. Centrifuge at 13,000g for 20 s.
5. Remove the cartridge insert from the tube and transfer the flowthrough to a microcentrifuge tube (see Note 5). Place the cartridge insert back into the tube.
6. Add 0.4 mL of cold (4°C) 1X wash buffer to the spin cartridge. Centrifuge at 13,000g for 20 s. Discard the flowthrough. Repeat this wash step three additional times (see Note 6).
7. Wash the cartridge twice with 400 pL of cold (4°C) 70% ethanol as described in step 6.
8. After removing the final 70% ethanol wash from the tube, centrifuge at 13,000g for 1 min.
9. Transfer the spin cartridge insert into a fresh sample recovery tube. Add 50 ||L of sterilized, deionized, previously preheated water to the spin cartridge (see Note 7). Centrifuge at 13,000g for 20 s to elute the cDNA. The procedure may be stopped at this point and the reactions stored at -20°C.
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