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. You can use OligoCalc
  • 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 25 nmol DNA oligo with 5' phosphorylated ends (click 5' Mods at the bottom) from IDT.

Note: if your oligo insert is large 40+ bp, use PAGE purification (it's a little slower and more expensive).

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

• Make oligos 100 µM
• 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.