Low yield with Ampure XP cleanup

[akbowser]

Hi all,
I am having some issues with my Ampure cleanup step and would love some troubleshooting help.

I'd say that every time I use the ampure beads (at 1.8 ratio) I lose about 50% of my product, which seems like A LOT. (I've tried my best to optimize the pcr conditions, but I think the biggest problem is that my template DNA is super crappy (literally - it is extracted from feces) and full of inhibitors.)

I'm already struggling to get enough ng of product made for NGS, so I'm pretty tired of losing it (and this high quality taq is expensive!) The beads expire in 2013. I make new EtOH every time. Might anyone have any idea about what could be going wrong??

[Rocketknight]

That's quite a lot of loss for one single-sided Ampure cleanup. Bear in mind, though, that Ampure XP cleanup will eliminate DNA under a certain size, so is it possible that the DNA loss you're measuring is just primers/dNTPs/small fragments being removed from the mix? What method did you use to quantify your DNA pre- and post-cleanup?

[pmiguel]

What is your assay method for determining your mass of DNA at each step? Could you give us a particular example? That is pick a typical sample and go through the yield you have at each step. (Make sure to give us total mass recovered at that step, not just the concentration.)

If you are initially assaying the amount of DNA using UV spectrophotometry, it is likely you are overestimating the amount of DNA you have. Generally >50% (and often >90%) of the absorbance at 260 nm of the typical genomic DNA prep derives from stuff that is not DNA. (RNA, degraded RNA, phenol, etc.)

--
Phillip

[kerplunk412]

The protocol says that overdrying the beads can significantly decrease yield, have you tried using a shorter drying time?

[docbio]

What about the expiration date? I am generally curious if people have any experience with "expired" Ampure XP beads? I have been inadvertently using some which are just a couple of months out of date and have ordered fresh, but I'm wondering what the real shelf life is, as they seem to still be performing fairly well.

[SarahNGS]

underdrying beads

Not getting all of the ethanol off the beads can also significantly reduce yields. make sure that you see small cracks and that the color lightens up. I have not had a problem with overdrying.