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Duplex Oligo & Ligation Protocols

Four cartoon scientist penguins in lab coats. Two wear goggles, holding a pipette and a petri dish. One has a computer, another a flask with blue liquid.

Duplex Oligo Ligation 

1) Resuspend oligos to 100 μM with ultrapure water 

Example: If 29.3 nmol of oligo, resuspend in 293 μl ultrapure water 

2) Phosphorylate and anneal each complementary pair (TOP & BOTTOM) of oligos: 

  • 1 μl top oligo (100 μM stock) 

  • 1 μl bottom oligo (100 μM stock) 

  • 1 μl 10x T4 PNK Buffer (NEB) 

  • 6.5 μl ultrapure water 

  • 0.5 μl T4 PNK 

= 10 μl Total Volume 

3) Incubate reaction in the thermocycler using a Phos and Anneal program: 

  • 37°C x 30 min 

  • 95°C x 5 min 

  • Ramp down to 25°C at a rate of 5°C/min 

4) Dilute phosphorylated and annealed oligo duplexes from step #3 at 1:100 with ultrapure water 

Example: 2 μl of oligos in 198 μl ultrapure water 

5) Ligate oligos into CUT & CIP'd plasmid backbone: 

  • 1 μl of digested & CIP'd plasmid backbone (at a min concentration of 50 ng/μl)  

  • 2 μl of 1:100 diluted oligo duplex from step #4 

  • 5 μl of 2x Quick Ligase Buffer (NEB) 

  • 2 μl of ultrapure water 

  • 1 μl Quick Ligase (NEB) 

= 11 μl Total Volume –> Incubate at room temperature for 10 minutes 

6) Transform the ligation reaction into competent cells

7) Recover competent cells in SOC 

8) Plate transformed bacteria and incubate O/N 


Learn how to design your construct and oligos for this type of cloning strategy: Duplex Oligo & Ligation Cloning


Need dilution calculations? Check out our online tools: Dilution Calculator

Test tubes with penguin illustrations as labels, aligned in a rack. Each penguin is cartoon-like with a black, white, and orange design. Stripe tube containing penguins.

Dephosphorylation of 5’ Ends of Backbone DNA using Quick CIP (NEB #M0525)  

1) Linearize backbone with restriction enzyme(s) 

2) To the digest tube, add 1 μl of Quick CIP (NEB) for every 1 pmol of DNA ends

3) Incubate at 37°C for 10 minutes 

4) Immediately proceed to gel purification of CUT & CIP'd backbone 

Note: Heat inactivation of enzyme(s) and Quick CIP is not necessary as long as gel purification immediately follows 10-minute Quick CIP incubation.

Critical: Final concentration of CUT & CIP'd backbone must be at least 50 ng/μl if the next step involves duplex oligo ligation.


Learn more about the molecular biology behind how ligation reactions work: Duplex Oligo & Ligation Cloning

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