older than 2 weeks enriched beads

[ilma]

Hei,

I would like to re-sequence library prepared 4 weeks ago. In the manual it is said that enriched beads should be sequenced within 2 weeks. Any observations about sequencing older beads? Thanks!
Ilma

[Aniki]

I am currently sequencing beads that are older than 2 weeks. I will tell you the outcome once I have the analysis. I have asked my FSA about this before and she said that they are good for two weeks and tend to degrade slowly past that. You might be pushing it a little for 4 weeks, as I have never waited longer than 18 days.

[MissDNA]

One time we were having problems with our machine and could not sequence. We had already enriched some beads, support recommended we should freeze the beads and thaw only for sequencing. I don´t recall the amount of time we left them frozen but they ended up sequencing very well.

[ilma]

Thanks for responses! I thought the beads should not be frozen. But if they do work after thawing then I might freeze them next time "just in case".
And yes, Aniki, let me know if the sequencing went fine.

[ajthomas]

They say that beads shouldn't be frozen, but apparently it doesn't kill them. The control beads come frozen and they sequence just fine. Also, a few weeks ago I absent-mindedly put the beads from a few reactions in the freezer after I finished the enrichment. Naturally I wasn't too happy with myself after finding them in the freezer, but they sequenced just fine.

[Aniki]

Hey I got sequencing results for over 2 week old beads. My FAS did not tell me to freeze it, I just left it in the fridge for over 2 weeks. Well run was good except the fact that only 370 000 raw wells were detected on each side of the plate. Which basically means that I lost half the data. Otherwise the run was not too shabby (~400bp average). Not sure if this is time in fridge related or the fact that the emulsion breaking kit I used was a bad lot.

Good luck.

[MissDNA]

Hey I got sequencing results for over 2 week old beads. My FAS did not tell me to freeze it, I just left it in the fridge for over 2 weeks. Well run was good except the fact that only 370 000 raw wells were detected on each side of the plate. Which basically means that I lost half the data. Otherwise the run was not too shabby (~400bp average). Not sure if this is time in fridge related or the fact that the emulsion breaking kit I used was a bad lot.

Good luck.

Which lot did you use? There are at least 2 lots that have been associated with low number of raw wells.

The person who told me to freeze the beads was from the old support group in Penzberg. I miss those guys.

[Aniki]

Which lot did you use? There are at least 2 lots that have been associated with low number of raw wells.

The person who told me to freeze the beads was from the old support group in Penzberg. I miss those guys.

I used lot 93866020. I achieved an abnormally low enrichment value at 2-3% even though my titrations were pretty decent. I have already voiced my concerns in the other thread.

[MissDNA]

I would blame the lot then and not the old beads for your problem.
We observed the same problem in one of our runs. I contacted support and after all they finally admitted that this lot has been associated with some other cases of low raw well number, and are replacing our bad run.