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  • ion torrent

    Hi all,
    I want to pre-amplify a 70Kb gene prior resequencing via ion torrnet. could any one please tell me what is the best way to do this.
    Thanks

  • #2
    When you say pre-amplify, from how many samples (and if more than one, how many runs do you want to pack it into)? Do you care only about exons or the entire gene?

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    • #3
      Originally posted by krobison View Post
      When you say pre-amplify, from how many samples (and if more than one, how many runs do you want to pack it into)? Do you care only about exons or the entire gene?
      I want to do this in about 50 samples and i am interested in entire gene.
      I think I can do it in one run using the 318 chip.

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      • #4
        One possibility would be to design (and validate!) a series of long PCR amplicons to tile the gene (each one overlaps the previous one by a bit).

        The pooled amplicons from each sample then generates a new fragment library. One thing to watch out for is that primer-derived sequences will have artificially high variant rates due to primer synthesis errors.

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        • #5
          There is a long lange pcr kit offered by Qiagen. this kit will generate a library of 8 overlapped amplicons (10 kb each) for my 70 kb gene. I think i can use it and then with biorubter or covaris I can generate fragments with 200bp lenght for ePCR and sequencing afterward. What do you think....

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          • #6
            Hi All,
            How can I ligate ion torrent barcodes to my DNA fragments.
            Regards,Tarek

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