NanoBio: Oligonucleotide Inserts

Design of oligonucleotide inserts
Note: if your oligo insert is large 40+ bp, use PAGE purification (it's a little slower and more expensive).
 * (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.
 * General idea: Create 2 oligonucleotides that contain your part flanked by the appropriate BioBrick/BioFusion sticky ends.
 * Design considerations. Make sure that:
 * either primer will not form a stable internal hairpin structure, &Delta;G < -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.

Hybridize ssDNA to form ds insert
to give 30 µL total volume
 * Note: this procedure is appropriate for 5' phosphorylated oligonucleotides only.
 * Make oligos 100 µM in distilled water.
 * 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


 * 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)
to give 30 uL total volume
 * 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
 * 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.


 * Back to Protocols.