Knight:Annealing and primer extension with Taq polymerase

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This protocol uses annealing and primer extension to generate a short fragment of DNA (~100 bp) using Taq polymerase. The DNA fragment can be immediately used in a TA cloning reaction. (To proceed to a restriction digest step, purification is necessary.)


  • Two oligos which overlap by ~20 bp.

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


  1. Dilute the two oligos to a concentration of 25 μM using H2O.
  2. Mix the following in a 0.6 mL sterile tube
    • 9 μL PCR supermix
    • 0.5 μL oligo 1
    • 0.5 μL oligo 2
  3. Anneal and extend the two oligos together by placing the mixture in a thermal cycler (MJ Research, PTC-200) and running the following protocol.
    1. 94°C for 5 mins
    2. 94°C for 30 seconds
    3. 56°C for 30 seconds (or whatever an appropriate annealing temperature is)
    4. 68°C for 30 seconds
    5. Repeat steps 2-4 2-3 cycles
    6. 68°C for 5 mins
  4. Use fresh 1μL PCR product in a TOPO TA cloning reaction.


  • For oligos greater than 50-60 bp in length, there can often be problems with errors or deletions in the primers. Therefore, it might be worth ordering your primers with an extra purification step such as PAGE. Invitrogen custom primers offers this service for an extra fee.


  1. Stemmer WP, Crameri A, Ha KD, Brennan TM, and Heyneker HL. Single-step assembly of a gene and entire plasmid from large numbers of oligodeoxyribonucleotides. Gene. 1995 Oct 16;164(1):49-53. PubMed ID:7590320 | HubMed [Stemmer-Gene-1995]