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