
Hello, I was following your protocol and precipitated the DNA with 95% ethanol, followed by centrifugation. I then tried to resuspend it using wash buffer, but found that the DNA could not be fully resuspended no matter how many times I pipetted it up and down—there was still a lot of u
Read moreChung-Te Chang Author Answered 7 hours ago
Institute of Biochemistry and Molecular Biology, National Yang Ming Chiao Tung University, Taiwan
That step is not meant to fully dissolve the DNA. As the protocol says, "Add 750 μL of wash Buffer to the DNA pellet and gently resuspend by pipetting until fully dispersed. Transfer the entire suspension to a miniprep spin column..." So the goal here is not complete dissolution, but just getting the pellet dispersed well enough to transfer. Since the buffer is still ethanol-rich, the DNA is not expected to fully dissolve at this step. The pellet just needs to be dispersed well enough for transfer onto the column. In our hands, the pellet does not need to be broken up very finely at this step. As long as the suspension can be transferred onto the column, we do not observe any difference in the final result. If transfer is difficult, the troubleshooting section says, "Fully disperse in 750 µL of wash buffer before loading; trim the pipette tip to ease transfer."
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