I am using the RPA III Ribonuclease Protection Assay Kit and am seeing aberrant migration with pointed or smeared bands. Can you help me troubleshoot?
If the supernatant from the final precipitation step is not completely removed, salts from the precipitation mixture will be concentrated and may lead to aberrant migration (“tunneling”) of the protected fragment. It is recommended that the final pellet be briefly respun after the supernatant is removed, and that the residual supernatant be thoroughly aspirated using a very fine-tipped pipet.