Bead Enrichment - infinite washes

[ajthomas]

Unfortunately my remaining breaking kits have lot number 93866020, which was not one recognized by the person I spoke with at Roche as having problems, but is listed in this thread.

I too will be interested in your results. Everything I had was either 93848420 or 93859020, so I called Roche and they replaced them all, but what they sent was 93866020. I've used that lot a couple of times now. It seems to be similar to 93859020, although to a lesser degree. I have to do a lot of washes, but by the end the beads seem okay. I plan to do the sequencing run on those beads tomorrow; I hope they work okay.

[HMorrison]

I experienced this yesterday and today; Lot number 93859020. Yesterday I was working with 3 samples each amplified (1xSV) using 1 and 3 cpb with Lib-A A. Today I was working with the same 3 samples amplified (2xSV) using 2cpb with Lib-A A. Final enrichment for *all* of the titration samples was around 10%. Today I called Roche, and didn't finish enriching. They will be replacing the kit. Unfortunately my remaining breaking kits have lot number 93866020, which was not one recognized by the person I spoke with at Roche as having problems, but is listed in this thread. I will setup one sample at 1 and 3 cpb as I had the first time and see what happens.

I will be very interested to know the results. Thanks.

[erikt]

I experienced this yesterday and today; Lot number 93859020. Yesterday I was working with 3 samples each amplified (1xSV) using 1 and 3 cpb with Lib-A A. Today I was working with the same 3 samples amplified (2xSV) using 2cpb with Lib-A A. Final enrichment for *all* of the titration samples was around 10%. Today I called Roche, and didn't finish enriching. They will be replacing the kit. Unfortunately my remaining breaking kits have lot number 93866020, which was not one recognized by the person I spoke with at Roche as having problems, but is listed in this thread. I will setup one sample at 1 and 3 cpb as I had the first time and see what happens.

[MissDNA]

Yesterday we had a meeting with our FAS and he said lot 93837120 was fine and we should keep using it, and no longer use 93848420 and 93852420. All the boxes we have in our lab belong to the lots I mentioned. Looks like there is a problem with much more lots than we originally thought.

[HMorrison]

Bead enrichment problem--picture

See total binding on first try.

Problem lots (bead recovery kit lot #):
93866020
93871620
93859020
93837120

[vlee2]

We had same problem with Bead Recovery Reagent LOT93859020. You need to call tech support and request replacement.

[HMorrison]

Aniki,
We've been seeing this problem a lot lately and the only solution seems to be to repeat the initial melt step. Will try to get more details. But the first time around, we would have calculated an 80-100% enrichment; it drops to 8-20% range (normal) on the second round. Seems like a lot of steps are not working the way they used to 2 yrs ago. We use LibA kits primarily, but have also experienced it with LibL.

[uhany]

Hi Aniki

White beads that actually covers the brown beads are no worth. They need to be washed off anyway. If you invert the MPC couple of times very slowly they will get dissolved in the supernatant and from where they can be removed very easily.

thanks

[mohdsos]

I had it once, I figured out that I had enrichment rate of 80% WOW. I think you need to check of primer dimer in case if it was amplicons. in general it must be copy number issue. do another emulsion with 70% less CPB, if you used 2 then use 0.5 cpb.

Mo

[Aniki]

Hey,

has anyone had issues with the enrichment step where the white beads literally ALL attract and stick to the brown beads. They congregate in a way where they are not in between the brown beads but instead coat the brown beads from the outside. Their attraction is relatively weak and the white beads break off very easily, however, this leads to quite a few washing steps (40+).

One thing of note is that the library I received was not very clean with both genomic and adapter contamination quite noticeably in a bioanalyzer.


If anyone has had this problem and a solution please give help!

Thank you so much.