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

Similar Threads
Thread Thread Starter Forum Replies Last Post
Rapid Library Library Quantitation using concentration not RFU GraemeFox 454 Pyrosequencing 9 10-21-2011 12:27 AM
Expected Library size after adaptors JPC SOLiD 3 10-04-2011 08:54 AM
Rapid Library Prep 454 HELP, HELP, HELP!!! Giancarlo Sample Prep / Library Generation 0 09-01-2011 09:34 AM
NEBNext 454 rapid library TonyBrooks Sample Prep / Library Generation 1 08-01-2011 01:56 PM
low cDNA concentration after the RAPID protocol dina 454 Pyrosequencing 0 07-11-2010 12:48 PM

Thread Tools
Old 02-15-2012, 07:50 AM   #1
Location: West Virginia

Join Date: Jun 2011
Posts: 14
Default 454 Rapid Library adaptors for NEBNExt protocol

Hi all,
We are examining cost cutting measures and considering moving to NEBNext Quick DNA Library prep. We have a 454 Junior and all of our kits and reagents are for rapid libraries. With NEBNext I have to order the adaptors separately. When I go to the IDT website, the only rapid library option for oligos includes the MIDs, which is fine. When I click on one of them, I tells me there is an Adaptor A and B. However, in the Roche rapid library protocol and the NEBNext protocol, when they refer to the adaptor ligation, it sounds like there is a single adaptor - not A and B. In the rapid library kits, there is a single tube of adaptor. The NEBNext protocol at the adaptor ligation step says to "add 1.0ul of adaptor to the reaction tube" with no mention of concentration. Am I supposed to mix MID adaptors we purchased from IDT in equimolar concentrations then add 1ul of that? At what concentration?

Thanks for any enlightenment.
Coyk is offline   Reply With Quote
Old 02-18-2012, 07:49 PM   #2
Senior Member
Location: Purdue University, West Lafayette, Indiana

Join Date: Aug 2008
Posts: 2,300

You need to anneal the oligos together to form a single Y adapter. Ligation of two (identical) Y adapters to either end of insert DNA yields your library molecules. These are strand denatured prior to amplification. Each strand will then have an "A" one one side and a "B" on the other. So the oligos really are "A" and "B".

Get the correct concentration and annealing protocol from IDT.

pmiguel 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 01:46 PM.

Powered by vBulletin® Version 3.8.9
Copyright ©2000 - 2018, vBulletin Solutions, Inc.
Single Sign On provided by vBSSO