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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.)



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. 55°C for 30 seconds (or whatever an appropriate annealing temperature is)
    4. 72°C for 30 seconds
    5. Repeat steps 2-4 2-3 cycles
    6. 72°C for 5 mins
  4. Use fresh 1μL PCR product in a TOPO TA cloning reaction.



Error fetching PMID 7590320:
  1. Error fetching PMID 7590320: [Stemmer-Gene-1995]