Go Back   SEQanswers > Applications Forums > Sample Prep / Library Generation

Similar Threads
Thread Thread Starter Forum Replies Last Post
TruSeq / SPRIworks? jlove Illumina/Solexa 10 07-23-2012 07:45 AM
TruSeq v3 Cluster Kits with TruSeq SBS kits? rgejman Illumina/Solexa 5 07-13-2011 10:33 AM
Truseq kathyglory Introductions 0 06-10-2011 01:39 AM
help with TruSeq upendra_35 Sample Prep / Library Generation 3 06-08-2011 11:27 AM
smallRNA v1.5 versus TruSEQ Fiji_surf Illumina/Solexa 0 05-02-2011 03:48 PM

Thread Tools
Old 06-19-2012, 06:50 AM   #1
Location: MI

Join Date: May 2012
Posts: 11
Default Let's Talk About TruSeq

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??
farrel75 is offline   Reply With Quote
Old 06-19-2012, 11:58 PM   #2
PhD Student
Location: La Jolla

Join Date: Jun 2012
Posts: 33

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.
SS00 is offline   Reply With Quote
Old 06-20-2012, 01:24 AM   #3
Senior Member
Location: London

Join Date: Jun 2009
Posts: 298

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We don't use Ampure beads that are out of date because we've found they don't keep so well.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.
TonyBrooks is offline   Reply With Quote
Old 06-20-2012, 03:16 AM   #4
Location: Ireland

Join Date: Sep 2011
Posts: 86

@TonyBrooks What size-selection problems have you had with the Truseq DNA kits? Do other kits use different size-selection methods?
Rocketknight is offline   Reply With Quote
Old 06-20-2012, 03:55 AM   #5
Senior Member
Location: London

Join Date: Jun 2009
Posts: 298

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.
TonyBrooks is offline   Reply With Quote

Thread Tools

Posting Rules
You may not post new threads
You may not post replies
You may not post attachments
You may not edit your posts

BB code is On
Smilies are On
[IMG] code is On
HTML code is Off

All times are GMT -8. The time now is 10:49 AM.

Powered by vBulletin® Version 3.8.9
Copyright ©2000 - 2020, vBulletin Solutions, Inc.
Single Sign On provided by vBSSO