DNA concentration with Ampure?

[jdp]

Hi all,
To reach reasonable DNA concentrations to put my pooled libraries on MiSeq I need to concentrate my samples i.e. my pooled libraries.

I have a huge range of DNA concentrations over the ~100 samples I am going to pool.

Through equimolar combination and dilution I will end up with 200ul of sample with a concentration at least 10x too low (around 0.2nM)!

Concentrating through SpeedVac is no option since my samples are in buffer and not in H2O.

I was thus planning to concentrate the pool with one step of Ampure beads and eluting them in 10x less volume. So I would elute 40ul from 200ul sample + 200ul bead to ideally rech a concentration of ~2nM.

Does anybody have experience with this? Would that work? How big of a loss do I have to expect?

Thanks a lot for your help!
jdp

[nucacidhunter]

10-20% loss is usual. If you have only one pool, it is more convenient to use a column. The ratio of bead is depend on your library size distribution. If clean libraries have been pooled, higher ratio of beads are safer. By using low ratio you might loose some of shorter library fragments.