EZ Bead

[quark]

Hi,
I´m working on SOLiD 5500 EZ Bead library amplification and have one big problem. All the amplified beads, either enriched or unenriched go to the waste container. Can anybody possibly help me finding the reason? Thanks very much.

[JPC]

Your beads really shouldn't end up there even if the emPCR is failing. What do you see in your enriched and un-enriched tubes? is there anything at all or are they completely empty? also, how does your filter look?

[quark]

Hello, both tubes (enriched, unenriched) are filled with transparent fluid, seems like watter, without anything inside. Considering the filter, it seemed to be empty, no magn.beads within. I tried to reuse it another day and some of mag. beads remaining in the Enricher Input tube easily passed it. On the contrary, the new filter worked properly, magn. beads taken from the Enricher Input Tube remained within. So what next? Is the filter supposed to be the key problem? Thanks for your answer.

[JPC]

After a cycle you should see at least a trace of beads on your filter, if you don't then I expect they aren't getting this far. Do you see anything in the QC output tube?

[quark]

After a cycle you should see at least a trace of beads on your filter, if you don't then I expect they aren't getting this far. Do you see anything in the QC output tube?

The QC output tube is also filled with transparent fluid without anything. And one more question,could the used filter (after enrichment) be altered to such degree, that it wouldn´t stop mag. beads one more time or is it simply not working from the begining?Thanks.

[JPC]

If you trace back the lines from the waste container you'll find one comes from the bottom of the filter, one each from the two mixer valves and two from the cleaning manifold. Are there any traces to suggest which route they are taking to get into your waste?

If the beads are going via the cleaning manifold then you probably have a problem with how you are setting up the machine, during a run only the cleaning line should be plugged into the cleaning manifold, is that how your set up looked?

[JPC]

The QC output tube is also filled with transparent fluid without anything. And one more question,could the used filter (after enrichment) be altered to such degree, that it wouldn´t stop mag. beads one more time or is it simply not working from the begining?Thanks.

I doubt it, but why would you want to reuse a filter anyway? AB should supply the correct amount for the number of emulsions you have.

[quark]

I doubt it, but why would you want to reuse a filter anyway? AB should supply the correct amount for the number of emulsions you have.

I checked the lines, but the fact is, that we cleaned the enricher, so no residual beads remained in none of them. So this way I can´t find it out. The reusal of the filter was ment only to check its properties "in vitro" outside the enricher, whether it worked properly during the proces. I´m wondering if its not a valve matter as far as there is no distribution of beads. And another fact is, that the solution from the B rack (CS sol1) seems to remain full, the Release Buffer around 40ml of 55ml and only half of Capture buffer has been spent. The same with solutions from rack A, they were not spent the whole amount, but I can´t tell you exactly as far as I have them no more. thanks.

[JPC]

Your rack A and B volumes sound normal if you have done a 'clean' but not a 'prime'...and even after a prime they won't be empty.

If your sample output and QC tubes have the right volumes and the rest of the tubes are empty then it sounds like the run was okay.

My best guess would be user error, after all it's very complicated! I would go back through the protocol in fine detail, ensure the capture bead tube was in place (and not the empty bottle that the Sip assembly come on) etc

[quark]

Thanks for your advices. Everything of the machine lines seems to be OK. Volumes within unenriched beads, enriched beads and QC tube OK, but no beads at all. On Monday I try to check the Enricher and find out where exactly the way of beads ends. I will appreciate consulting it with you. Have a nice weekend.

[snetmcom]

occasionally the filter actually "breaks" and allows beads to pass through. The result is all the beads in the waste.

This can happen from bad filters or from overloading the filter with beads. Supposedly the last update helped with this.

[quark]

Hello, so today I have run an enrichment one more time with a new filter...and it was OK. The mag. beads could be found within the filter and the bottle of enriched beads was full of them, few beads could be seen at the bottom of the QC tube. Some went to the bottle for unenriched beads, but it doesn´t matter at all. And the waste was empty!!! I hope that I won´t find out that only this one was working and the rest of them are "broken". As far as it is the same lot and part number I´m not able to detect the wrong ones. Maybe I could replace them with new ones ordered separately from other consumables supporter. Thanks everybody for your experiences and help.

[quark]

Hi, one more time. I thought, that it was OK, but it is not. Another enrichment run was unsuccesfull. I think the filter is being stucked with polystyrene beads, so that the magnetic beads cannot pass through (this time there were traces of mag. beads on filter, but only at the edge of it; the amount of emulsion was within the range). Before (once when it worked) I entered the weigh number, that was within the range 117g (for E80), but the real weigh of emulsion was higher by 29g (146g)... and the filter worked, the mag. beads were separated and not in the waste as in this case. So can it be a matter of software (more diluted sample can be separated) or what. thanks for your answers.

[JPC]

Hi quark, if your emulsion is over 118g then something has gone wrong, I would start again if this was one of my experiments.

Are you saying you put all 146g into the machine and told the software it was 117g? and what was it about the run that was 'unsuccessful'?

JPC

[quark]

[QUOTE=JPC;73046]Hi quark, if your emulsion is over 118g then something has gone wrong, I would start again if this was one of my experiments.

Are you saying you put all 146g into the machine and told the software it was 117g? and what was it about the run that was 'unsuccessful'?

The run that was succesfull was only a test run with mag. beads purified from waste and put back into the input bottle. The fluid from the unenriched bottle was poured into the input bottle, too (but because of misunderstanding the final weigh of the "emulsion" was by 29g higher). And it worked. The regular runs with regular amount of emulsion ended with mag. beads in the waste.