Knight:Annealing and primer extension with Klenow polymerase

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This protocol uses annealing and primer extension to generate a short fragment of DNA (~100 bp). The DNA fragment is prepared for cloning by restriction digest.


  • Two oligos which overlap by ~20 bp and have restriction enzyme sites at the 5' ends as in the diagram below. See Restriction Digest for notes on cutting near the ends of linear DNA fragments.

Oligo 1:    5' ---RE site-------------------------------- 3'
Oligo 2:                                        3' --------------------------------RE site--- 5'

Calculating amount of oligo for reaction

This should be checked for errors -Reshma 19:03, 12 May 2005 (EDT)

[math] \rm{X\ L\ oligo} = \frac{\frac{Y\ g\ oligo}{(330\ g/mol\ of\ nt)(W\ nt/oligo)}\ mol\ of\ oligo}{Z\ mol/L\ oligo\ stock}[/math]


  1. Dilute the two oligos to a concentration of 10 or 25 μM using H2O
  2. Mix the following in a 0.6 mL sterile tube
    • 10 μL 10X restriction enzyme buffer
    • 1 μL 100X BSA
    • X μL oligo 1 (typically 1 μg or more)
    • Y μL oligo 2 (typically 1 μg or more)
    • (87 - X - Y) μL deionized sterile H2O
  3. Anneal the two oligos together by either placing the mixture in a thermal cycler (MJ Research, PTC-200) at 94°C for 5 mins, a cool down for 0.1°C/sec to 65°C, 65°C for 5 mins, then a cool down for 0.1°C/sec to 37°C. Alternatively, the tube can be placed in a beaker of boiling water and let cool to room temperature.
  4. Add 1 μL Klenow 3'[math]\rightarrow[/math]5' exo- polymerase to mixture.
    Vortex polymerase before pipetting to ensure it is well-mixed.
  5. Add 1 μL dNTPS (equal to 0.25 mM final concentration of each dNTP).
    Recommend using a thermal cycler for the following incubation steps.
  6. Incubate 1 hr at 37°C.
  7. Heat inactivate polymerase by incubating at 75°C for 20 minutes.
    See Restriction Digest for more information on the following steps.
  8. Add 1 μL restriction enzyme(s) to mixture.
  9. Incubate for a minimum of 2 hrs.
  10. Heat inactivate restriction enzyme by incubating at 80°C for 20 mins.
  11. Purify DNA as necessary


W. P. Stemmer, A. Crameri, K. D. Ha, T. M. Brennan, and H. L. Heyneker. Single-step assembly of a gene and entire plasmid from large numbers of oligodeoxyribonucleotides. Gene, 164(1):49–53, 1995. PubMed