In the initial denaturation step, complete denaturation of the DNA template at the start of the PCR reaction is essential. Incomplete denaturation of DNA will result in the inefficient use of the template in the first amplification cycle and, consequently, poor yield of PCR product.
The annealing temperature may be estimated as 5°C lower than the melting temperature of the primer-template DNA duplex. If non-specific PCR products are obtained in addition to the expected product, the annealing temperature can be optimised by increasing it stepwise by 1-2°C.
Usually, the extension step is performed at 72°C and a 1-min extension is sufficient to synthesise PCR fragments as long as 2 kb (kb = kilobase = 1000 bp). When larger DNA fragments are amplified, time is usually extended by 1 min per 1000 bp.The number of PCR cycles will basically depend on the expected yield of the PCR product.
After the last cycle, samples are usually incubated at 72°C for 5 min to fill in the protruding ends of newly synthesised PCR products.

PCR procedures: conditions for the
reaction mixture
Contamination of the DNA must be prevented by:
! Separating the areas for DNA extraction andPCR
! Using sole-purpose laboratory equipment
! Autoclaving and aliquoting
! Adding a control reaction

Some useful tips:
• DNA extraction and PCR reaction mixing and processing should be performed in separate areas.
• Use of sole-purpose vessels and positive displacement pipettes or tips for DNA sample and reaction mixture preparation is strongly recommended.
• All solutions, except dNTPs, primers and Taq DNA polymerase, should be autoclaved. Where possible, solutions should be aliquoted in small quantities and stored in designated PCR areas.
• A good practice, to confirm absence of contamination, is to add a control reaction without template DNA.

PCR procedures: components

Many PCR machines are now available in 48-, 96- or 384-well formats. This, combined with the use of multichannel pipettors, can greatly increase the number of reactions that can be done simultaneously. If several reactions need to be simultaneously prepared, a master mix should be used as follows: water, buffer, dNTPs, primers, MgCl2 and Taq
DNA polymerase in a single tube. This will then be aliquoted into individual tubes.
Considerations:
Template DNA. Nearly any standard method is suitable for template DNA purification. An adequate amount of template DNA is between 0.1 and 1 μg for genomic DNA for a total reaction mixture of 100 μl. Larger template DNA amounts usually increase the yield of non-specific PCR products.
Primers. (1) PCR primers should be 10-24 nucleotides in length. (2) The GC content should be 40%-60%. (3) The primer should not be self-complementary or complementary to any other primer in the reaction mixture, to prevent primer-dimer and hairpin formation.(4) Melting temperatures of primer pairs should not differ by more than 5°C, so that the GC content and length must be chosen accordingly. (5) The melting and annealing temperatures of a primer are estimated as follows: if the primer is shorter than 25 nucleotides, the approximate melting temperature is calculated with the formula: Tm = 4(G + C) + 2 (A + T). (6) The annealing temperature should be about 5°C lower than the melting temperature.
MgCl₂ concentration. Because Mg ²⁺ ions form complexes with dNTPs, primers andDNA templates, the optimal concentration of MgCl2 has to be selected for each experiment. Too few Mg ²⁺  ions result in a low yield of PCR product, and too many will increase the yield of non-specific products. The recommended range of MgCl₂ concentration is 1 to 3 mM, under the standard reaction conditions specified.
Taq DNA polymerase. Higher Taq DNA polymerase concentrations than needed may cause synthesis of non-specific products.

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