ProceduresPrimerExtension < Lab

---+ Primer Extension or Oligo Overlap Extension
To stitch together large DNA templates from oligonucleotide fragments.

---++ Using Klenow Fragment (3&prime;&rarr;5&prime; 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&times; | 2 µl |
| dNTPs | 2 mM each | 0.3 |
| Klenow Fragment (3&prime;&rarr;5&prime exo–) | 5,000 units/ml | 0.2 µl |
| ddH<sub>2</sub>O |  | 13.5 µl |
| *Total* | *&nbsp;* | *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

   1 [[http://www.neb.com/nebecomm/products/productm0212.asp][NEB website]]

Barrick Lab > ProtocolList > ProceduresPrimerExtension

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