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 µM	2 µl
Oligo 2	10 µM	2 µl
NEBuffer 2	10×	2 µl
dNTPs	2 mM each	0.3
Klenow Fragment (3′→5&prime exo–)	5,000 units/ml	0.2 µl
ddH₂O		13.5 µl
Total		20 µl

First mix together oligonucleotides and water. Heat to 90°C 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 37°C for 10 minutes to 1 hour.

References

NEB website

Topic revision: r2 - 2012-05-31 - JeffreyBarrick