SPRI beads - over vs under drying

[SeqTroubles]

Hi All,

Just wondering what your experiences are in regards to over vs under drying SPRI beads during NGS.

I am currently using Nextera XT with Ampure XP beads. I give a 5-10min RT dry but I still get very low yield 1-4ng/ul in my libraries.

Do you feel if I extended this to 15mins as per protocol I may get better yield? How do I know if I have over dried the beads?

[nucacidhunter]

You could normalize the volume and run PCR amplicons before and after clean up on Bioanalyser and look for quantity of DNA in the selected size range to estimate recovery.

I consider beads dry when they start to lose shine and just a crack starting. Having said that, there are protocols like PacBio and 10x that ask only for 1-2 min drying before elution and Ethanol droplets are clearly visible.

Over-dried beads will have lots of cracks or scale look. Longer incubations will increase recovery but the difference might be very small which will not justify for most applications.

[thermophile]

Under dry and you can carry a little EtOH into your next step. Over dry and your DNA may not elute off the beads. If you think you are losing DNA at this step, you could increase your binding time not your drying time.

[Carcharodon]

A little bit of EtOH carry-over is preferable to loss, which can happen if you over-dry your beads. And that is a very, very fine line.

I would actually reduce your dry times to below 5 minutes (try 2 minutes), and see if there's improvement.

This won't work for getting rid of EtOH from the beads themselves, but a trick for getting extra EtOH out of the tube and off the walls is to take a clean, dry, sterile wooden toothpick, and dab the ethanol away. It works like a charm.