Hybridization washing

[Tim]

Hello group,
I need some suggestions.

I have sepharose beads with ca. 1 million copies of covalently bound
single stranded DNA (same sequence). They are mixed with beads that
DON'T have any single-stranded DNA.
I need to count the ratio "how many beads contain DNA".

I'm hybridizing detection oligos (ca. 20 bases) to the single-stranded
DNA. At the moment I'm washing 4x with 20 mM Tris pH 7.5, 5 mM MgAc2
after the hybridization step. Then I analyze them by FACS.

I would like the difference between positive (carries DNA ->
fluorescent) and negative (no DNA, no fluorescence) to be as clearly
separated as possible. At the moment my two bead populations are
overlapping a little bit. Can someone suggest more stringent washing
conditions?

I'd go for
- formamide
- tween 20
- others?

Concentrations? I'd like to wash at room temperature, to keep it simple
for now. I'm washing the beads by resuspension/centrifugation, so I'd
like to keep the number of washes within limits as well.

Thanks for any useful advice!

Tim