---+ 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 | | ddH<sub>2</sub>O | | 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 1 [[http://www.neb.com/nebecomm/products/productm0212.asp][NEB website]]
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Topic revision: r2 - 2012-05-31 - JeffreyBarrick