no enrichment of beads --> any ideas why?

[lichtfaengerin]

Hi,

working with the 454 FLX system using the Lib-A Kit (adapted it for unidirectional sequencing). Up to now I had no problems with getting beads to sequence but since my last sequencing project one year passed. Now I'm not sure if have the problems concerning the enrichment because the beads expired approx. a year ago. Does anyone of you has experience with expired DNA Capture Beads?

Up to the step of Enrichment I do don't have any problems. I can imagine that it's a problem with the DNA Capture beads as I get something which looks like beads which can not be centrifuged (I do get a bead pellet but in addition this greasy bead layer (or what so ever))

Or can it be a problem of too short DNA fragments? (156bp)

Any ideas?

Thanks a lot for your help!

Cheers
Lisa

[uhany]

lisa,

Expired DNA capture beads worked fine with me. There is a modified protocol (pcr volume and cycle) in my454 for amplicons <200bp, I think you should try that but that probably needs using libL kits. In my opinion emulsion recovery is critical with the amplicons concentration which we calculate from the product size and the product amount. Be careful in putting the right amount of amplicons in the emPCR and should work all right. thanks