Cloudy eluates with AMPure XP beads?

[osl]

Hi all,

I was trying to pool my ChIP DNAs into one for library construction using the AMPure XP beads (300 ul sample + 1.6x beads).
However, after adding 31 ul of TE for elution, I recovered around 37 ul AND it looks a bit cloudy.
I did try repeating the XP purification once more (37 ul of eluates + 1.6x beads) but got similar results.
I suspect the cloudiness is from the PEG-containing buffer of the beads, which somehow hasn't been efficiently removed.

My main question is whether this "cloudiness" would interfere with later steps in library prep (next step would be Ends Repair) or it'd be better to remove it by MinElute.
Any inputs on how to avoid this are also very welcome.

Thank you!
osl

[Isequencestuff]

What magnet are you using? It seems like your volume might be a little high for efficient bead binding, hence causing the cloudiness. Could you pool, then aliquot out into smaller volumes?

[kcchan]

How much ethanol are you using for your washes? If you have that much sample you may need up to 1mL of 80% ethanol in order to wash off all the PEG in the tube.

[osl]

For magnet, I was using the one from Millipore (Magna GrIP Rack -- 8 well), which wasn't a very effective magnet. I did try with the stronger Invitrogen one later. It seems a bit better but still had the same issue.

That's true, I should have used more ethanol. I just used 200 ul of 80% EtOH in the washes. But as I've mentioned, in my 2nd attempt, which I only had 40 ul of sample, the eluates still looked a little bit cloudy.

One thing I forgot to mention is the beads have past the expiray date, which is Jun 2012. I did test it right beforehand and it worked well (>90 recovery by Qubit). But that time, I did not vortex the beads before use. Compared to this, that run had much less beads and did not have the cloudy issue.

Anyway, do you guys have good experience with the Ampure XP beads in general? Do you think new beads would be free of this problem?
Also, what do you usually do or recommend when pooling multiple ChIP DNAs? For now, I'll go back to use MinElute.

Thanks again!

[silin284]

contamination in DNA could cause that

I have seen that if the DNA has a lot of secondary metabolite. i work with plants so it is pretty common. you can clean up the dna using a column before ampure xp. we perfer to do a phenol chloroform instead. best wishes...

[osl]

Wow, may be that's why! The ChIP DNAs are from Arabidopsis. Also, the test sample that worked fine had gone through a Qiaquick column beforehand.
Thank you for the input!