NanoBio: Overlapping Oligonucleotide Inserts

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Overlapping Oligonucleotide Inserts

  • General Idea: To create medium sized inserts ~85-150 bp, four overlapping oligonucleotide inserts can be used. The region of overlap between the first antisense oligonucleotide and the second sense oligonucleotide should be ~5-6 bp, as if the overlap region were sticky ends from a restriction digest.
  1. Especially if you are making a sequence which is repetitive on the amino acid level, use Gene Designer to back-translate into an optimized DNA sequence.
  2. Add the 5' and 3' ends to your insert, then break this into four fragments.
    1. The first fragment will be 5'-CTAGA (first half of your insert).
    2. The second oligo will be 5'- complement of (first half of your insert + 6 bp)-T.
    3. The third oligo will be 5'-
  • CAjoF 12:35, 26 January 2008 (CST):