Let's Talk About TruSeq

[farrel75]

Hi everyone,
My lab and I are in the process of taking our maiden voyage on the MiSeq and we are trying to make the process as cost effective as possible. We are considering having me do the library prep rather than have it sent to the core facility. We need more than the 12 barcodes we get from the A kit but we don't need the entire B kit.
So the question is: What about TruSeq? Can we keep it in the fridge for an extended amount of time? Is it a one time use thing? Any suggestions??
Thanks!

[SS00]

Do you mean storage of the kit? You can store the kit in the freezer (not the fridge!) until the expiry date which is written on the top of the box.

If you are not going to use the whole kit at once you need to aliquot End Repair Mix, A-tailing Mix, and all the Controls (if you use them) into single use aliquots to avoid freeze thaw.

Once you have done this, it should be fine to revisit your kit as many times as you need.

[TonyBrooks]

We used a TruSeq RNA kit that was 9 months passed use-by date and it still produced excellent libraries. We followed SS00's advice and aliquoted the reactions out into smaller volumes when the kit arrived and stored everything at the recommended temperature (written on each individual tube).
We don't use Ampure beads that are out of date because we've found they don't keep so well.
There are also alternative prep kits that are supplied in smaller pack sizes which may be more suitable if you don't want the hassle of aliquoting everything out (Bioo, NEB). Personally, I'm not so keen on the TruSeq DNA kits as size selection is a real problem. The TruSeq RNA kits are excellent though.

[Rocketknight]

@TonyBrooks What size-selection problems have you had with the Truseq DNA kits? Do other kits use different size-selection methods?

[TonyBrooks]

We've never gotten the post ligation gel purification to run properly. Libraries run too slowly with EthBr and too fast with SYBR. A 400-500 excision either gives us libraries >600bp or 300-400bp. We could probably optimise it, but we don't have the time.
We didn't see this problem with the older Illumina PE kit or with NEB kits. I also think, the latest NEB kit has a protocol for gel-free selection, giving libraries ~ 350bp. It's not as tight as gel urification, but takes minutes, not hours.