Making dsDNA Inserts from Oligonucleotides

Goal:

Create dsDNA with desired sticky ends from oligonucleotides

Design of oligonucleotide inserts

  • (Optional)Use Gene Designer from DNA2.0 to create an optimal DNA sequence from a protein coding sequence. This is particularly helpful if you are making a sequence which is repetitive on the amino acid level. You can download this program for free here.
  • The procedures below are well suited for making dsDNA inserts that range in size from 15-100bp in length. If the desired insert is shorter than 15bp, consider using site-directed mutagenesis. If the desired insert is longer than 100bp, PCR, denovo synthesis, or Gibson assembly are better options.
  • Design considerations. Make sure that:
    • either primer will not form a stable internal hairpin structure, ΔG < -3 kcal/mole. You can use OligoCalc
    • either primer does not contain a restriction enzyme that you will be using in subsequent steps, e.g. EcoRI, PstI, SpeI, XbaI, or NotI for Biobricks cloning or BsaI for Golden Gate cloning
  • Order 25 nmol DNA oligo with 5′ phosphorylated ends (click 5′ Mods at the bottom) from IDT using ebuy.
  • Note: if your oligo insert is large 40+ bp, use PAGE purification (it’s a little slower and more expensive

Hybridization of ssDNA to form a dsDNA Insert

Note: this procedure is appropriate for 5′ phosphorylated oligonucleotides only.

  • Dissolve oligonucleotides to 100 µM in distilled water.
  • Mix:
    • 3 uL 100 µM sense oligo
    • 3 uL 100 µM anti-sense oligo
    • 3 uL 10 x PNK buffer (NEB catalog # B0201S, available in common buffer stocks)
    • 3 uL 0.5 M NaCl
    • 18 uL distilled water

to give 30 µL total volume

  • Place in boiling water bath for 2 min., then remove water bath from the heat source and allow the DNA (still in the water bath) to cool to room temperature.
  • Alternatively, use a thermocycler to heat the solution to 100C for 2 min, then ramp down to 25C over 75 m.
  • 1x PNK buffer (NEB catalog # B0201S) contains: 70 mM Tris-HCl, 10 mM MgCl2, 5 mM Dithiothreitol, pH 7.6.

Phosphorylation of 5′ ends & hybridization (only if you didn’t order 5’phosphorylated oligos)

  • Note: it is really just better in terms of time & efficiency to order 5′ phosphorylated oligos. Really.
  • Mix:
    • 3 uL 100 µM sense oligo
    • 3 uL 100 µM anti-sense oligo
    • 3 uL 10 x PNK (polynucleotide kinase) buffer
    • 2 uL 10mM ATP
    • 2 uL T4 polynucleotide kinase (PNK)
    • 17 uL distilled water

to give 30 uL total volume

  • Incubate at 37C for 1.5 hours.
  • Add 4 uL 0.5 M NaCl.
  • Place in boiling water bath for 2 min., then remove water bath from the heat source and allow the reaction (still in the water bath) to cool to room temperature.

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