Molecular Biology: A Practical Guide - Velikov V.A. 2013

PCR
Performing DNA PCR amplification

PCR is carried out in a volume of 10-50 µl. The working concentration of primers in the reaction mixture is 0.2-1 pM/µl. The amount of template DNA added to the reaction ranges from 10 ng (plasmid DNA) to 1000 ng (genomic DNA). The working concentration of Jag polymerase is

0.01-0.05 units/µl. The number of cycles is 30. The rate of DNA extension by Taq polymerase during PCR is estimated to be 1000 NUCLEOTIDES per minute.

The optimal primer concentration is often determined empirically (for calculation, see Lab 6.3). It is not recommended to use more than 50 pM per sample, otherwise non-specific annealing and primer-dimer formation may occur. Typically, the thermal cycler is programmed as follows:

1. T_d = 95 ^oC, 1 min or more - initial template Denaturation, one step

2. 25-30 PCR cycles:

T_d = 95 ^oC, 10 s or more - denaturation

T_a = 50-65 ^oC, 30 s or more - primer annealing

T_e = 72 ^oC, 1 min or more - elongation, extension

3. T = 72 ^oC, 1 min or more - final extension, one step

4. T = 4 ^oC - storage mode

As an example, the PCR Amplification protocol for the mitochondrial NADH dehydrogenase Gene of Nikolsky's viper is presented (Velikov et al., Bulletin of Saratov State Agrarian University, 2006, No. 3).

Materials and Equipment

Thermal cycler, total DNA of Nikolsky's viper Vipera nikolskii (40 ng/µl), primers (10 pM/µl), dNTP mix (2 mM), magnesium chloride solution (25 mM), Taq polymerase (5 units/µl), 10x PCR buffer.

Solutions

- Reaction mixture. Preparation of the mixture in a final volume of 20 µl:

1. 10x PCR buffer (200 mM Tris-HCl, pH 8.4; 500 mM KCl; 0.01% Tween 20) - 2 µl;

2. 2 mM dNTP mix (a mixture of all 4 deoxynucleoside triphosphates at a concentration of 2 mM each) - 2 µl;

3. 25 mM MgCl₂ solution - 2 µl;

4. Primer ND2 For: 5'-GCATTTTCATGACCACCACC-3' - 2 µl;

5. Primer ND2 Rev: 5'-GAGTGAGGGGTAAGATAGTG-3' - 2 µl;

6. Target DNA: total DNA of Nikolsky's viper (40 ng/µl) - 3 µl;

7. Deionized Water - 6.8 µl;

8. Taq polymerase - 0.2 µl.

Procedure

1. In a sterile 0.5 (0.2) ml thin-walled plastic tube, mix the above components and bring the volume up to 20 µl with deionized water. Add the enzyme last and immediately transfer to -20°C!

2. Centrifuge for 15 s. Apply a small amount of mineral oil on top of the reaction mixture to prevent evaporation (approx. 30 µl using a pipette or a drop). If the thermal cycler is equipped with a heated lid, adding oil is not necessary. Place the tubes into the thermal cycler.

3. Run the following PCR program: initial template denaturation at 95°C for 5 min, followed by 30 amplification cycles: 95°C for 30 s, 57°C for 30 s, 72°C for 1 min, then a final extension step at 72°C for 5 min (final strand completion), and hold at +4°C for storage. The calculated annealing Temperature for both primers is 60°C; the actual temperature is set 3°C lower to ensure efficient annealing and subsequent primer extension.

4. Perform DNA Electrophoresis and gel imaging (for the electrophoretogram, see Appendix 11; amplicon size is 911 bp). In Real-Time PCR, electrophoresis is omitted, and the process is monitored via fluorescence.