Primer Extension or Oligo Overlap Extension

To stitch together large DNA templates from oligonucleotide fragments.

Using Klenow Fragment (3′→5′ exo)

This reaction will extend to single-stranded oligos with overlap to generate a complete double-stranded DNA.

The complete reaction looks like this:

component [stock] volume
Oligo 1 10 M 2 l
Oligo 2 10 M 2 l
NEBuffer 2 10× 2 l
dNTPs 2 mM each 0.3
Klenow Fragment (3′→5&prime exo) 5,000 units/ml 0.2 l
ddH2O   13.5 l
Total   20 l

First mix together oligonucleotides and water. Heat to 90C for 2 minutes and cool at room temperature to denature and anneal the strands for extension. Add buffer and dNTPs. Mix. Add Enzyme. Mix gently. Incubate at 37C for 10 minutes to 1 hour.


  1. NEB website
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Topic revision: r2 - 2012-05-31 - 14:39:45 - Main.JeffreyBarrick
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