Overview

Short to medium length (up to 120 bp) fragments of dsDNA can be generated directly from two complementary single-stranded oligonucleotides. Custom overhangs for ligation or In-Fusion can be introduced as well, simply by having some non-complementary ends. The two single-stranded Oligos then need to be annealed into a double-stranded fragment. The idea is to heat up the mixture to close to 100 C and let it slowly cool down to room temperature over the course of one hour.

Materials

• Thermocycler
• 10x annealing buffer, final (1x) concentration: 10mM Tris-HCl, 50mM NaCl
  • closest match is NEB Buffer 2 (1x: 10mM TrisHCl, 50mM NaCl, 10mM MgCl₂, 1mM DTT)

Procedure

Mix (30µl total volume, 20µM final DNA concentration):

• 15 µl sterile ddH20
• 6 µl sense oligo 100µM
• 6 µl antisense oligo 100µM
• 3 µl NEB buffer 2 (10x)

Incubate on thermocycler:

• 2 min @ 98C
• 60 cycles:
  • 1 min, decreasing temperature by 1.3 C per cycle
• Cool to 4C

Adapted from [http://openwetware.org/wiki/PrbbBB:Oligo_Annealing OpenWetWare]