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LongAmp Taq DNA Polymerase Guidelines |  | | LongAmp Taq DNA Polymerase Guidelines |
Protocol
- 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). - 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. - 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. - Mg++ and additives:
The final Mg++ concentration in the 1X reaction buffer is 2 mM. This gives satisfactory amplification to 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. - 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. - 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. - 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. - 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:
4-10°C - Cycle number:
Generally, 25-35 cycles give optimal amplification. Up to 45 cycles may be required to detect low-copy targets.
References:
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.
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