Library prep question - ligation of P1 and P2 adapters

[rbstruijk]

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]

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

[JPC]

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]

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:doi%2F10.1371%2Fjournal.pone.0009320

JPC

[rbstruijk]

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