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.docStandard PCR from ABgene.
PCR is a widely used technique with many applications. What follows is a basic protocol for PCR, although reaction conditions, such as times and temperatures of incubations and concentrations of thermostable DNA polymerase, MgCl2, primers and template DNA will vary and need to be optimised for different experiments.
It is important that the optimal MgCl2 concentration is determined empirically by testing in the range 1.5-4.0 mM MgCl2 for each primer set (see PCR Optimisation Plate). Reduced efficiency or non-specific products may result if insufficient or excess MgCl2 is used. For samples that contain EDTA or other metal ion chelators, the MgCl2 concentration in the reaction mix should be raised proportionately. MgCl2 concentrations should also be adjusted in parallel with high concentrations of sample DNA and dNTPs.
The ideal length of single-stranded DNA primers is between 15-30 bases. Primer sequences should not complement within themselves or with each other, especially at the 3' ends. The GC content of the primers should be approximately 50%. Where possible, primers should be designed to incorporate G or C at the 3' end. Ideally, the melting temperatures of the primers should be within 5°C of each other.
1) Use a thin-walled plastic consumable free of DNases and RNases.
Reagent Volume Final Concentration
10X reaction buffer (without MgCl2) 5 µl 1X
20 mM dNTP mix 2 µl 0.2 mM of each nucleotide
25 mM MgCl2 3 µl 1.5 mM
Primer mix (10 µM each) 2.5 µl 0.5 µM each
Template DNA 1-2 0µl 1-250 ng (dependent on source)
Thermostable DNA Polymerase (5 units/µl) 0.25 µl 1.25 units
Double-distilled, sterile water to 50 µl
2) Mix the reagents in the tube or plate well. If a thermal cycler without a hot lid is being used, the reagents should be overlaid with 50 µl of Mineral Oil.
3) Close or seal the tube or plate well and spin briefly to ensure all the reagents are collected at the bottom.
4) Start the reaction with a DNA denaturation step (1-5 minutes at 94°C).
5) Perform 30-40 cycles of PCR:
Denature +94°C for 20 s
Anneal 50-65°C for 30 s ()
Extend 72°C for 60 s
For the extension step start with 60 s/kb. In general, higher annealing temperatures yield more specific products. A good starting temperature is 5°C below the melting temperature of the primers. To determine the optimum, test at 5°C increments (or smaller increments) until the maximum specificity is reached. DNA with a high GC content may require a very high (> 60°C) annealing or denaturation temperatures. Alternatively, 7-deaza-dGTP, mixed with dGTP, or DMSO can be added to reduce hydrogen bonding between complementary bases, thus reducing the effective annealing/denaturing temperatures required. The half-life of Taq DNA polymerase (30 minutes at 95°C) suggests that a denaturing temperature of 96°C should not be exceeded.
6) After the last cycle, incubate for a further 10 minutes at 72°C to complete the extension of both strands. After PCR, the samples should be stored at 4°C (short term) or -20°C (long term).
7) The amplification products can be analysed by agarose gel electrophoresis and visualised using ethidium bromide staining.