[Rocketknight]

Hey, sorry to dredge this thread up again, but I have another question! Illumina recommend a double-Ampure cleanup after ligation with a 1:1 ratio of Ampure. This confused me way back when I started this thread, but now I realize that as well as cleaning up the reaction this is also a way of removing DNA below about 140bp - adapters and adapter dimers, in other words.

So here's my next question: Can I save time by doing a different cleanup at this step (e.g. QIAQuick PCR Purification) if I'm going to do downstream exome capture? I'm assuming that the exome capture step will be largely unaffected by the adapter dimers. Or have I overlooked something?

[Heisman]

Small amounts of adapter dimers will probably not be too damaging, but how much time do you really save by doing a QIAQuick PCR Purification over an Ampure XP Bead cleanup? It can't be more than 5 minutes. Is that worth deviating from the protocol when you are going to do a capture for probably at least 24 hours?

[Rocketknight]

It's just me being lazy, but two consecutive Ampure cleanups take about an hour and a half with Illumina's recommended incubation and drying times, compared to about ten minutes to column-purify.

But yeah, you're right. If there's any uncertainty I'll stick with the Ampure method.

[Heisman]

I don't know what Illumina recommends, but it's probably unnecessary. Mix beads with sample, vortex vigorously, 5 minute incubation, pellet beads with magnet, discard supernatant, add in 70-80% EtOH (enough to cover the beads), discard the EtOH, repeat the wash, discard the EtOH, suck out residual EtOH at the bottom of the tube with a fine-tipped pipette, place tube in heat block for a few minutes, add in water/TE to elute, vortex and incubate for 2 minutes, then transfer out. Whole process never takes me more than 20-25 minutes if I'm trying to get done quickly and only am working with a few tubes.

Another idea (that I have not tried) is to mix in the beads for the first cleanup, do the vortext/5 minute incubation step, discard supernatant, do one wash with 70-80% EtOH, discard the EtOH, and then directly add in the beads for the second cleanup and continue from there.

[Rocketknight]

Awesome, but do you lose much DNA from the shorter Ampure incubation time? I feel like not all of it would have precipitated after 5 minutes.

[Heisman]

I don't lose more DNA, I don't think. You can test this very easily of course.

[theduke]

So here's my next question: Can I save time by doing a different cleanup at this step (e.g. QIAQuick PCR Purification) if I'm going to do downstream exome capture? I'm assuming that the exome capture step will be largely unaffected by the adapter dimers. Or have I overlooked something?

I just tried this, but the adapter carryover was way too high. The Qiagen columns have a size cut-off of around 70 bp, so I'm not sure its the way to go post-ligation. I have cut the Ampure clean-up to just a single round though because I don't want to lose too much mononucelosomal DNA (this is for ChIP-Seq) and that looks perfectly fine on HS DNA chips, especially when you combine it with Ethanols pre-size selection PCR to convert the Y-shaped adapters.