LongAmp™ Taq DNA Polymerase Guidelines


  1. Reaction setup:
    We recommend assembling all reaction components on ice and quickly transferring the reactions to a thermocycler preheated to the denaturation temperature (94-95°C).
  2. Template: 
    The quality of the DNA template is essential for long-range PCR amplification. Recommended amounts of DNA template for a 50 μl reaction is as follows:
      up to 15 kb above 15 kb
    genomic DNA 1 ng - 500 ng 10 ng - 1 µg
    plasmid or viral DNA 1 pg - 1 ng 10 pg - 10 ng
    Successful amplification above 20 kb largely depends on the quality of DNA templates and the primer sequences.
  3. Primer:
    Primers are generally 20–40 nucleotides in length and ideally have a GC content of 40-60% (2). Computer programs such as Primer3 (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi) can be used to design or analyze primers. For amplicons larger than 20 kb, it is desirable to have primers with GC content above 50%, matched Tm above 60°C, and primers at least 24 nucleotides in length. The final concentration of each primer in a typical PCR reaction is 0.05-1 μM, ideally 0.2-0.5 μM.
  4. Mg++ and additives:
    The final Mg++ concentration in the 1X reaction buffer is 2 mM. This gives satisfactory amplification of most amplicons. However, Mg++ can be further optimized in 0.2 mM increments using MgSO4.
    For some difficult targets such as GC-rich sequences, additives such as DMSO (3) and formamide (4) may be included to improve amplification.
  5. Denaturation:
    An initial denaturation of 30 seconds at 94-95°C is sufficient for most amplicons from pure DNA templates. For difficult templates such as GC-rich sequences, longer denaturation of 2-4 minutes at 94-95°C is recommended prior to PCR cycling to fully denature the template. With colony PCR, an initial 5 minute denaturation at 94-95°C is recommended. Subsequent denaturation can be done between 10-30 seconds.
  6. Annealing:
    The annealing step is typically 10-60 seconds. Annealing temperature can be optimized by doing a temperature gradient PCR starting 5°C below the calculated Tm.
  7. Extension:
    Extensions are recommended to be carried out at 65°C. For amplicons below 20 kb, extension can be carried out from 60-68°C. A final extension of 10 minutes at 65°C is recommended.
  8. 2-step PCR
    When primers with annealing temperature above 60°C are used, a 2-step PCR protocol is possible.
    Initial denaturation: 
      94-95°C 30 seconds
    25-45 cycles: 
      94-95°C 10 seconds
      60-65°C 50 seconds per kb
    Final extension: 
      60-65°C 10 minutes
    Final soak: 
  9. Cycle number:
    Generally, 25-35 cycles give optimal amplification. Up to 45 cycles may be required to detect low-copy targets. 


  1. Barnes, W.M. (1994) Proc. Natl. Acad. Sci., 91, 2216–2220.
  2. Foord, O.S. and Rose, E.A. (1994) PCR Methods Appl., 3, 149–161.
  3. Sun, Y. et al. (1993) Biotechniques, 15, 372–374.
  4. Sarkar, G. et al. (1990) Nucleic Acids Res 18, 7465.