NanoBio: Oligonucleotide Inserts

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.
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, 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).

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

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.

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.