Knight:Annealing and primer extension with Taq polymerase

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Revision as of 21:30, 14 June 2006 by Reshma P. Shetty (Talk | contribs)
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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.
  • RS 21:30, 14 June 2006 (EDT): I recently have been trying to make a promoter via this method. I sequenced 3 different clones and all of them had a 1bp deletion in the same position. This was despite the fact that I had ordered my primers to be PAGE purified. I emailed Invitrogen and they said that they will resynthesize and ship me a new primer free of charge. We'll see what happens.


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