Ampure XP beads drying

[tchiang]

Does anyone know if drying out the beads till it's cracked actually affects the elution efficiency?

I am following a library prep protocol and they say to dry the beads for 15 minutes after the alcohol step. I noticed the beads are cracked and are dry by 8 minutes.

[nucacidhunter]

Drying time is dependent on temperature, plastic ware and amount of bead. For instance, drying time for the same amount of bead in a 1.5 ml tube would be different from a PCR plate well. The best indicator for dry bead is lack of shininess and just one crack. Over drying where bead has multiple cracks can reduce elution efficiency. To maximise elution in such cases, after adding elution buffer incubate at RT at least for 5 minutes and mix frequently to wet the beads.

[Baseless]

We have good experience drying them in a PCR cycler at 37°C till a visible crack in the pellet can be seen. Usually like 3-5 mins instead of 15.

[WhiteSeal]

We have been using AMPure XP beads for the Illumina TruSeq DNA/NANO/Amplicon prep for some time now and we always use 1.5ml LoBind tubes and dry them for 5 minutes in a heat block at 37celcius. With this protocol we have had no issues.

[kerplunk412]

I have done a few experiments to test this exact question, and I have also probably purified over a thousand samples with Ampure. I don't think over drying actually effects the ability of the DNA to elute from the beads, I think it just makes the beads very clumpy so that DNA on the inside of a clump is not exposed to elution buffer. As long as the beads are resuspended very well for elution there is not much trouble with over-drying, in my experience.

For efficient elution from possibly over-dried beads the method that nucacidhunter recommended sounds perfect to me. Also, you can tell if beads are not full resuspended if they fall to the bottom of the tube instead of staying in solution during the elution incubation.

[tchiang]

Thanks for the replies. I am doing a 5 min incubation at RT after adding elution buffer. Hopefully I will be getting everything out of the beads.

[WhiteSeal]

Just on a different note, does everyone wash their bead pellets while on the magnet? (I do btw)

I have read on the MinIon forum that someone was removing the samples off the magnet, then wash with alcohol thourougly -> use magnet to create pellet and then remove the alcohol (this done twice) and thus receiving a much higher yield?

Anyone any experience with that?

[h11826]

When I wash the beads with EtOH, I usually just do it on the magnet, though it sometimes disrupt the pellet if I pipet directly to the pellet. I don't see a
significant difference. My colleagues do their washes either way and I don't think it matters.

[Baseless]

@WhiteSeal: Back in my early FLX days, I had some complete losses of libraries while removing the pellet from the magnet for washing. It might be a bit of voodoo, but since I avoid disturbing the pellet on the magnet during washing like the plaque we rarely lost any libraries except every now and then when we had to take a chance on sacred 'low input' samples.