Sanger sequencing

Magazine

Are you contaminating your DNA on cleanup columns?

A very frequent scenario when cleaning up your samples (plasmids or PCR products) for DNA sequencing is to load your DNA to silica-based membrane in spin columns. Unfortunately some columns (even those of established manufacturers) may not have a really favorable shape and consequently your DNA gets dirty during purification.

The penultimate step of the cleanup procedure is spinning empty columns, a minute or two, to dry them out. Then an elution buffer is added and after another brief centrifugation samples are collected and shipped for sequencing. But they may still contain some ethanol or salts from buffers used during purification. These should have been removed when centrifuging empty columns but recommended spin times are usually too short and due to the shape of spin columns some liquid remains in the cup and is removed not sooner than at the elution step. As a result, your sample gets contaminated.

You can confirm this easily. Proceed as described in the protocol but just before elution transfer the inner cup into a clean eppendorf tube and spin empty for 5 min at recommended speed. In most cases you will see a small volume of some liquid at the bottom of the collection tube, most likely ethanol (up to 10 µl). This liquid will inhibit your sequencing reaction.

What can be done here? The simplest way is to extend the centrifugation time to approx. 3–5 min when drying. But there are inner cups with internal lip holding small (dead) volume of liquid when centrifuged in angle rotors. It is therefore recommended to centrifuge empty tubes for 2 min to dry them out and then, without removing the tubes from the rotor, rotate the inner cup 180º and spin again for additional 2 min. This way any liquid trapped inside should be removed. Make sure the inner cup is completely dry and you cannot see any traces of liquid and transfer it to the elution tube. Proceed with elution as recommended by the kit manufacturer.

 

Richard Nádvorník, richard.nadvornik@seqme.eu

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