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-   -   Library prep question - ligation of P1 and P2 adapters (http://seqanswers.com/forums/showthread.php?t=20063)

rbstruijk 05-15-2012 06:08 AM

Library prep question - ligation of P1 and P2 adapters
 
Hi there,

I'm trying to grasp how the nick translation works exactly after ligating the P1 and P2 adapters to A-tailed DNA fragments. Does the reaction at 72oC exchange the nucleotides of the insert DNA (5' to 3') or does it go in the other direction, only exchanging the nucleotides in the adapter sequences towards the 'outisde'? This is very important to us since we want to bisulfite-convert our libraries after ligation. Can anyone shed light on this?

rbstruijk 05-15-2012 06:09 AM

Above question is about the Solid 5500 single-ended library prep

JPC 05-15-2012 07:15 AM

After ligation wouldn't there be 4 nicks? 2 on each strand? if that is the case then it seems that which ever direction the reaction has the insert will be exchanged?

JPC 05-15-2012 07:27 AM

There's a diagram in this paper (from Life Technologies) that suggests that only the adaptors are affected (and also that I am wrong about the '4 nicks' thing.......)

http://www.plosone.org/article/info:...l.pone.0009320

JPC

rbstruijk 05-15-2012 11:52 PM

Thanks JPC, it looks like only the adapters are in fact nick translated.


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