NanoBio: Oligonucleotide Inserts

Design of oligonucleotide inserts

• The sense oligo should be of the form:
  5' CTAGA(coding sequence)ACTAGTAGCGGCCGCTGCA 3'
  The bolded sequences will be the overhanging "sticky" ends.
• The anti-sense oligo should be of the form:
  5' GCGGCCGCTACTAGT(reverse complement of coding sequence)T 3'
• Design considerations. Make sure that:
  • either primer will not form a stable internal hairpin structure, ΔG < -3 kcal/mole
  • either primer does not contain a EcoRI, PstI, SpeI, XbaI, NotI, or BstEII site outside of the flanking regions
  • the insert for the forward primer does not begin with TC (or else a DAM I site (GATC) is formed, the A is methylated and XbaI cannot cut at its site)
• Order 100 nmol DNA oligo with 5' phosphorylated ends and PAGE purification from IDT.
• Note: if you're feeling stingy or in a rush and your oligo insert is small ~30 bp, you can skip PAGE purification and the insert will still likely be okay. For longer inserts, PAGE purification is highly recommended.

Hybridize ssDNA to form ds insert (start here if you ordered phosphorylated oligos(right move!))

• Mix:
  • 3 uL 100 µM sense oligo
  • 3 uL 100 µM anti-sense oligo
  • 3 uL 10 x PNK (polynucleotide kinase) buffer (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.
• 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.