Ampure XP size selection problem

[saw9]

Hello All,

I'm having a problem with size selection optimization using AMPure XP beads. Basically, I can't get any size selection regardless of volume added to my DNA. I've been using 1ug of 100 bp ladder in 28 uL H2O or Tris buffer and adding 0.7-1.2 volumes beads at .1x increments. The attached (ugly) gel image shows that there is really no size selection going on. Our protocol calls for a 6 minute incubation at RT but mine went a bit over. Still, I would expect to see some signs of size selection. Any ideas?

FYI, for the samples on the left, I used stock beads, while those on the right are 5X diluted in equal volumes of 40%PEG and 5M NaCl. The library prep protocol I use also has a size selection step that uses diluted beads in 1 part 5M NaCl, .6 parts 40% PEG and .4 parts H2O. At those concentrations, I DO get good size selection expected for each volume.

[pbluescript]

Does your ladder already have a loading dye added to it or do you add it after the size selection?

[saw9]

added after size selection

[luc]

Saw9,

I am just guessing, but it could be that a high concentration of DNA leads to the stronger than expected precipitation.
I observed it my own tests with a ladder which was considerably more concentrated than advertised. I guess at high DNA concentrations the size selection fails.

Which paper/protocol suggests your replacement buffer ?

[Isequencestuff]

Have you tried titrating below 0.7X volume AMPure beads?

[saw9]

Thanks for your replies. I also wondered about DNA concentration. I haven't tried titrating below .7x yet, but considering the reduction in PEG concentration it might look more like the results I get with the replacement buffer.

The protocol I'm using is from Wang et al. 2011, A low-cost library construction protocol for Illumina-based strand-specific multiplex rna-seq. PLoS One 6(10) e26426. doi:10.1371/journal.pone.0026426

It's been slightly modified from the original paper and I've made a few changes based on the enzymes we have available on hand. I can share if you like. Also, I beleive the author is a member here.

[saw9]

I should also mention that the replacement buffer (.6 parts PEG .4 parts H2O) is only used at one step in the protocol. The rest use equal volumes of PEG and NaCl where buffer replacement is a result of diluting the beads. Thanks again.

[SarahNGS]

My money's on too much DNA. Try quantifying your ladder via fluorescence. Or just dilute the DNA 10x and repeat the experiment.

[AndyG]

Saw9 how did the size selection go?

I'm using the same protocol and compared bead ratios of 1.2x and 0.7x and saw a decent difference, but I also realise that I will need to titrate further to totally remove any trace of smaller fragments.

[jwfoley]

What's your ladder?

I also had this problem, and as far as I could tell it turned out to be because there's something weird about certain brands of DNA ladder that makes them behave unusually with SPRI. http://seqanswers.com/forums/showthread.php?t=31787

You could also just use someone else's data to calibrate your size selection. This isn't exactly unexplored territory.