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  • #31
    Dear Phillip,
    I'm quite conscious on the SOLiD mate pair library, the steps and the structure of the mate (p1-insert-mpl-mpr-insert-p2) .. it's about one year I'm trying to improve the robustness of the method. Belong to my experience the protocol of the mp isn't satisfactory and mainly replicable.

    SO, I'm trying to find other way to overcame some steps that in my opinion are limiting. The circularization is one of these points. The problem to circularize DNA is that the IA (in the 5500 xl is formed by mpr and mpl) contains part of the ligation site for the annealing in sequencing. Therefore, the importance of knowing the IA sequence ( and it is irreplaceable).

    I was interested in that post, because you (or someone else) wrote the IA sequence with a string of NNNNN. I thought that my sanger sequence on the IA was with a double signal (2 picks) because of the NNNNN string.
    I'm not sure if it's more clear my question on the Mpl Mpr sequence!

    thanks for your answers

    chiara

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    • #32
      Hi chiara,
      I do not think there should be any NNNNN in SOLiD mate pair adapters. But you would need to explain what you mean by:

      I've tried to sequence the IA by Sanger technologies
      What exactly did you sequence? That is what did you prime your sequencing reaction with and what were your results?

      --
      Phillip

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      • #33
        Oo , I am also curious about the MPL and MPR adaptor, esp it contains a "mysterious" blocking oligo during the circulization step according to the protocol, is there anybody can tell me their "tricks"???


        thanks a lot!

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        • #34
          hello yksikaksi, I noticed you knew many about the SOLiD, so I think maybe you can help me out of my problem: do you know the sequence of the sequencing primer complemented with the P1 adapter, I want to know which of the chains in the dsDNA was binded to the beads, if you know this, could you please reply as soon as possible, thank you very much.
          Nothing is impossible!

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          • #35
            Originally posted by dongyongdong View Post
            hello yksikaksi, I noticed you knew many about the SOLiD, so I think maybe you can help me out of my problem: do you know the sequence of the sequencing primer complemented with the P1 adapter, I want to know which of the chains in the dsDNA was binded to the beads, if you know this, could you please reply as soon as possible, thank you very much.
            Dear dongyongdong,

            Perhaps you could have a look on this post of this thread.

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            • #36
              Dear yksikaksi,
              thanks for your quick reply, and I finally make myself clear about this queation, the P1adapter is fixed on the beads, so the sequence of the 5'-end of the P1 was what I want. Thanks again. best regards.
              Nothing is impossible!

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              • #37
                Please tell me the sequence of the adapter of MPL and MPR of Mate-Paired.
                I would also like to have a biotin position.
                I want to ligate another adapter to blunt-end after ligation the MP adapter.
                I'm worried biotin will drop from the adapter by blunt-end.

                thanks

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