Complete library prep degradation during -20C storage?

[Jerry Glenn]

Has anyone experienced complete library prep degradation during -20C storage? If so, was the cause ever determined? Illumina RNA-Seq libraries isolated with Invitrogen size select E-gels and stored at -20C for 6 months in our lab completely degraded.

[sehrrot]

Haven't experience yet but I felt a library kept -20C somtimes looks having a bit lower concentration than before I kept it in -20.

Actually Truseq library prep kit recommends "no more than one week storage between procedures". I really wonder whether this mention is for "degradation problem" or not. In my mind, it shouldn't be degradaded as it is just a nucleotide.

[pmiguel]

Hi Jerry,
Any chance this was a "Frost Free" freezer you had the samples in? Also, what assay did you use to determine that the library had "completely degraded"? Bioanalyzer chip or something else? Also, what does "completely degraded" mean, specifically?

--
Phillip

[gogreen]

I have the same questions as pmiguel too, What do you mean by 'totally degraded' in the context? . I've sequenced libraries which were stored for over 3 months and everything was fine. I've seen a drop in concentration after long term storage many times. But they are never totally vanished.

@pmiguel, How would a frost free freezer make a difference?

[aushev]

@pmiguel, How would a frost free freezer make a difference?

quoting the wiki, "In laboratories, self-defrosting freezers must not be used to store certain delicate reagents such as enzymes, because the temperature cycling can degrade them."

[heidurlo]

I had these very same thoughts. I use the Nextera XT kit and the recommended storing time in -20 freezer is only one week, which seems to be a very short time.
Are there any other people out there with stories of degraded libraries after a few weeks-months in the freezer?

[Genohub]

Yes, how you measured the "complete degradation" would be good to know. If your library concentration is really low or diluted that could be a cause. I've also heard of DNA sticking to tubes. Low bind tubes or tween should help with that.

- Genohub