General PCR Guidelines:
General PCR Guidelines:
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Template:
Use of high quality, purified DNA templates greatly enhances the success of PCR. Recommended amounts of DNA template for a 50 μl reaction are as follows:
|
DNA |
Amount |
|
genomicDNA |
1 ng–1 μg |
|
plasmid or viral |
1 pg–1 ng |
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Primers:
Oligonucleotide primers are generally 20–40 nucleotides in length and ideally have a GC content of 40–60%. Computer programs can be used to design or analyze primers. The final concentration of each primer in a reaction may be 0.05–1 μM, typically 0.1–0.5 μM.
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Mg++ and additives:
This protocol supports satisfactory amplification of most amplicons. However, Amplification of some difficult targets, like GC-rich sequences, may be improved with additives, such as Mg++ , DMSO or formamide.
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Denaturation: An initial denaturation at 95°C is sufficient for all amplicons from pure DNA templates.
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During thermocycling a 15 second denaturation at 95°C is recommended for most amplicons.
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Annealing:
The annealing step is typically 60 seconds. Annealing temperature is based on the Tm of the primer pair and is typically 60-72°C. Annealing temperatures can be optimized by doing a temperature gradient PCR.
Using Harborgen 2x SuperMerase Mix, 2-step protocol fit most amplicons. However, 3-step PCR protocol work also.
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Extension:
Extension times are generally 1 minute per kb. A final extension of 5 minutes at 72°C is recommended for routine PCR.
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Cycle number:
Generally, 25–35 cycles yields sufficient product. Up to 45 cycles may be required to detect low-copy-number targets.
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PCR product: The PCR products generated using Taq DNA Polymerase contain dA overhangs at the 3´–end; therefore the PCR products can be ligated to dT/dU-overhang vectors.
References:
1. Saiki R.K. et al. (1985). Science. 230, 1350-1354.
2. Powell, L.M. et al. (1987). Cell. 50, 831-840.
3. Sun, Y., Hegamyer, G. and Colburn, N. (1993). Biotechniques. 15, 372-374.
4. Sarkar, G., Kapelner, S. and Sommer, S.S. (1990). Nucleic Acids Res.. 18, 7465.