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  • MissDNA
    replied
    Ours was suppposed to happen this month but was postponed to January. I talked about amplicons a couple of months ago with a Roche representative and he said FLX+ was still not recommended for amplicons, and since no new bulletin or manual was released I am assuming it is not.
    Last edited by MissDNA; 12-07-2011, 02:04 PM.

    Leave a comment:


  • pmiguel
    replied
    Originally posted by MissDNA View Post
    Is FLX+ already validated for amplicon experiments? Last I heard, it was not.
    Good point. I don't know. (Our upgrade did not happen yet.)

    Elad,
    You might be better off just doing 2 shorter amplicons with FLX chemistry.

    Anybody using FLX+ for amplicons?


    --
    Phillip

    Leave a comment:


  • MissDNA
    replied
    Is FLX+ already validated for amplicon experiments? Last I heard, it was not.

    Leave a comment:


  • pmiguel
    replied
    You can probably get away with un-purified primers.

    About purifying the amplicons: I would use every method at your disposal to select against primer dimers. Cutting them out of a gel and then double AMPure purifying them is not unreasonable. (You can do it after pooling to reduce the number of samples you have to work with.)

    I think the problem is two-fold:

    (1) There is a high fold difference in the amplicon size (800 bp in your case) vs the primer dimer size (~80 bp?). That is 100x. So even a barely visible primer dimer is a problem when considered on a molar level.

    (2) Primer dimers can anneal back to full length amplicons after a denaturation, right? I mean, there is nothing to stop them doing so. Even after a size selection you will still have some glomming on to what you purify.


    So, those 2 factors combined work against a 454 amplicon experiment because the primer dimers are small enough to hide unnoticed among your products. If you get enough of them, they cause problems given that they will PCR amplify so much more easily than your intended amplicons.

    This effect would be exacerbated with FLX+ runs, I would expect.

    --
    Phillip

    Leave a comment:


  • eladSeq
    started a topic 454 GS FLX+ long reads question

    454 GS FLX+ long reads question

    Hi,

    I am a newcomer to deep sequencing and wanted to get some feedback on my project. My library consists of ~18,000 variants of an 860 bp gene on a plasmid. Because of its length I'm planning to use the 454 GS FLX+ system (which should have reads up to 1000 bp). I was also informed that I must use the one-way sequencing design with the Lib-L chemistry for getting the long reads and 1 million reads on a full plate.

    Therefore my plan is to prepare two amplicons using PCR with the Lib-L primers, one with the A primer on the 5' end, and one with the A primer on the 3' end, since I want reads from both ends. Does anyone know if these primers must be purified (HPLC of PAGE)?

    Also, is it always necessary to purify the amplicons with the AMPure XP beads, or will a zymo column purification suffice? Especially if I don't see adapter dimers on an agarose gel or BioAnalyzer run.

    Thank you,
    Elad

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