Ampure XP beads that do not stick to wall of plate

[dtm2451]

Hello,

I am currently having an issue that I have never seen before with my ampure bead purifications. The beads are not sticking sufficiently to the sides of my wells, and an inconsistent but significant fraction are coming out with the supernatant. I have personally been performing SPRI purifications on a regular basis for over a year and haven't changed anything reagent or plastics-wise that I think should cause the issue. I think the problem might be static electricity. Has anyone else ever had this problem before? And if so, how did you go about fixing it?

Thank you,
Dan

[JanaSEQ]

Hi Dan,
I am having the same issue but unfortunately I don´t know, how to fix it. Like you, I have been used Ampure purification for a long time and this problem occured recently.

[nucacidhunter]

If the beads are not past expiry date I would contact supplier/manufacturer Techsupport.

[dtm2451]

Thanks for the suggestion. I called tech support last week. TS said they have never had a case like mine where the beads would not bind tubes at all in the first step (1st DNA binding step, before the first sup removal). They suggested changing plastics, switching magnets, and checking the bead collection without any sample.

Only adding beads without sample led to proper binding. Thus the problem should be sample related, but that doesn't make sense to me either. My samples were a PCR reaction that I and other labs run all the time:

KAPA HiFi HotStart ReadyMix @1x
Primer (SMARTer amplification primer IIA)
cDNA (that had been run through a successful SPRI rxn just beforehand)

Thus I am still uncertain about the cause of the problem.

[dtm2451]

Hi Janaseq,

It's very annoying when these things happen in protocols that you feel like an expert with isn't it? I think our issues might be slightly different, however, because your binding issues come later. In my case, I wasn't even able to make it to the ethanol cleanup step at all without losing half my beads in the initial supernatent. I have some thoughts on the issue you are having, but I'll post them on your thread.

[Manna]

I have encountered this problem. Once the beads are pulled to the side of the tube, remove the original supernatant / binding buffer, but leave some behind. Leave behind whatever you need to so as not to disturb the beads. Add as much 80% ethanol as you can, and again pull the beads to the side. You don't have to take out every last drop of supernatant. I find that as I change out buffer for more ethanol, the beads stick better to the side.