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

Join Date: Jun 2011
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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.
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Old 02-18-2012, 07:49 PM   #2
pmiguel
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Location: Purdue University, West Lafayette, Indiana

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Default

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.

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Phillip
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