Silver: Oligonucleotide Inserts

Design of oligonucleotide inserts

• The sense oligo should be of the form:
  5' CTAGA(coding sequence)ACTAGTAGCGGCCGCTGCA 3'
• 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.

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

• 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.

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

• 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.