SEQanswers

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



Similar Threads
Thread Thread Starter Forum Replies Last Post
scaffolding GAII paired-end library with Hiseq mate-pairs stevebaeyen Bioinformatics 17 02-27-2013 01:45 AM
Agilent automated library preps-Low yields after pre-hybridisation PCR debbiehughes Sample Prep / Library Generation 0 11-21-2011 06:06 AM
Why and how are gDNA contaminations in RNAseq library preps bad? lukas1848 Sample Prep / Library Generation 1 11-04-2011 03:51 AM
SPRI-TE Illumina library preps GW_OK Sample Prep / Library Generation 5 08-23-2010 09:13 AM
Automating library prep for 454/GAII 454newbie Sample Prep / Library Generation 10 02-16-2010 08:39 PM

Reply
 
Thread Tools
Old 09-22-2009, 07:35 AM   #1
nickb
Junior Member
 
Location: London

Join Date: Sep 2009
Posts: 2
Default Ligase for DIY GAII library preps

Hi,
I've recently switched away from Illumina supplied to DIY library preps. I've been using the NEB Quick ligation kit (M2200) to ligate on my adapters. I've recently discovered this is 2,000 units/ul. The Illumina protocol uses 1 unit/ul (5 units in total). We have seen some unexpected chimeric reads in these PE libraries, where read 1 is a different chromosome to read 2. I suspect the excess of ligase maybe the reason. Although others I've spoken to are using this 2000 x ligase and seem to find it ok. What liagases are other people using? Has anyone had problems with inserts ligating to each other rather than the adaptors?
Cheers, Nick
nickb is offline   Reply With Quote
Old 09-28-2009, 08:02 AM   #2
elaney_k
Member
 
Location: Dublin, Ireland

Join Date: Mar 2008
Posts: 55
Default

Hi Nick,

AFAIK the Illumina protocol does actually use 5ul of the quick ligase from NEB (M2200) which is 2000U/ul, where did you get info to say it was 1 U/ul?
elaney_k is offline   Reply With Quote
Old 09-29-2009, 01:26 AM   #3
nickb
Junior Member
 
Location: London

Join Date: Sep 2009
Posts: 2
Default

Hi Elaney,

I have an early version of the RNAseq protocol (from before illumina sold kits for mRNA libraries) this states 1 unit / ul of DNA ligase.
I've had a look at some patents - one version I found here: http://www.faqs.org/patents/app/20090088327

The ligation step is here:
Step 5) Ligation
[0270]Materials: [0271]A-tailed genomic DNA (from Step 3) [0272]Quick ligase buffer (2) (NEB, B2200S) [0273]Annealed adaptor (15 μM) (from 4.) [0274]Quick Ligase (1 U/μl) (NEB, M2200L) [0275]PCR purification kit columns (Qiagen, 28104)

I think Illumina has just made a mistake with the Ligase concentrations on protocols and patents, as the Quick Ligase from NEB is 2000 U/ul.

Cheers, Nick
nickb is offline   Reply With Quote
Old 09-29-2009, 02:48 AM   #4
elaney_k
Member
 
Location: Dublin, Ireland

Join Date: Mar 2008
Posts: 55
Default

Ah! I had the same protocol and questioned Illumina tech support about it but never got a proper answer as it was an unsupported protocol at the time, I couldn't find any ligase from NEB to match the 1U/ul concentration so assumed it was a misprint.
elaney_k is offline   Reply With Quote
Old 03-05-2010, 10:36 AM   #5
BearClaw
Junior Member
 
Location: Boston

Join Date: Feb 2010
Posts: 9
Default

I'm sure this has been mentioned somewhere else at seqanswers, but I thought it was relavant to this thread.....NEB has a kit containing all the reagents for sample preparation except for adaptors and primers. Catalogue number E6000. I'm thinking that this is quite a bit cheaper than the Illumina kit, even if you have to order the HPLC-purified oligos and adaptors (with the phosphorothioate or phosphate additions where needed) from your favorite oligo supplier.
BearClaw is offline   Reply With Quote
Reply

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 11:53 AM.


Powered by vBulletin® Version 3.8.9
Copyright ©2000 - 2019, vBulletin Solutions, Inc.
Single Sign On provided by vBSSO