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Standard PCR Protocol

Standard PCR Protocol

Taq polymerase is a thermostable DNA polymerase named after the thermophilic bacterium Thermus aquaticus from which it was originally isolated by Chien et al. in 1976. Its name is often abbreviated to Taq Pol or simply Taq. It is frequently used in the polymerase chain reaction (PCR), for it is able to withstand repeated heating to 95 °C without significant loss of activity, an efficient method for greatly amplifying the quantity of DNA templates.

Prepare DNA template:
Usually, for plasmid DNA, 1-10 ng; for genomic DNA, 50-500 ng per reaction is needed. Normally, DNA template does not need to be purified. However, both purity and the amount of template can strongly influence the outcome of the reaction. So, it’s necessary to confirm the concentration and A260/A280 ratio of DNA template in advance.

Design primer:
Generally, primers used are 18-23 mer in length. Use Primer3 free online software or other assistant tools like Primer Premier to design proper primers. Choose the best primer pairs from those options according to the parameters, like size, TM, GC%, etc.

Determine annealing temperature:
Melting temperature (Tm) of primers can be calculated by the following formula: Tm = [(#of A + T residues) x 2] + [(#of G + C residues) x 4] °C. Tm-5 °C is a good annealing temperature to start with. However, optimal annealing temperatures can only be determined experimentally for a certain primer/template combination. Temperature gradient PCR is often a way to finalize an optimal annealing temperature.

Prepare 10x PCR reaction buffer, include:
100 mM Tris-HCl (pH 8.3)
500 mM KCl
15 mM MgCl2
0.1% gelatin
Note: The MgCl2 concentration is typically 10-15 mM. However, the optimum concentration needs to be determined experimentally. Mg2+ forms a soluble complex with dNTP's which facilitates dNTP incorporation, and stimulates polymerase activity. It also promotes and stabilizes primer and template interaction. Thus, increasing the magnesium concentration has the same effect as lowering the annealing temperature. Too low Mg2+ leads to low yields (or no yield) and too much Mg2+ cause nonspecific products.

For a 100 μl reaction, add:
10x PCR buffer 5 μl
DNA template Y μl
Forward Primer 1 μl
Reverse Primer 1 μl
dNTPs Mix 1 μl
Taq DNA Polymerase 0.25-0.5 μl
Sterile ddH2O X μl
Total reaction volume 50μl
1. Primers diluted to working concentration (10µM working stocks are sufficient for most assays). 2. Final Concentration of primers should be 0.1-0.5 µM.
3. Final Concentration of dNTPs Mix should be 200µM.
4. Final Concentration of DNA template should be 200 pg/µL
5. MgCl2 will depend on the system being utilized.
6. Mix gently by vortex and briefly centrifuge to collect all components to the bottom of the tube.
7. For some PCR machines that do not have a heated lid, mineral oil needs to be added to each reaction to prevent evaporation of the sample. The amplified DNA can be evaluated by agarose gel electrophoresis and subsequent ethidium bromide staining. Mineral oil overlay may be removed by a single chloroform extraction (1:1), recovering the aqueous phase.
8. Prepare a control reaction with no template DNA and add more sterile water to the same reaction volume.

A typical PCR program may be:
Initial denaturation, 4-8 min at 94-95 °C.
Denaturation, 15 sec at 94-95 °C.
Annealing, 15 sec at x °C (depends on Tm).
Extension, x sec (depends on product length, 1 min kb-1) at 72 °C.
Return to step 2 for 30-35 additional cycles.
Final extension, 10 min at 72 °C.
Keep sample at 4 °C until loading.
Notes: The amplification parameters will vary depending on the primers and the thermal cycler used. It may be necessary to optimize the system for individual primers, template, and thermal cycler.

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