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DNA Template

Amplification of templates with high GC content, strong secondary structure, low concentrations, or which produce products greater than 5 kb, may require adaptation of the following parameters:

• Use high quality, purified DNA templates.
• Approximately 10E4 copies of target DNA are required to detect product in 25-30 PCR cycles.
• Use 1pg–1ng of plasmid or viral templates.
• Use 1ng–1µg of genomic templates.
• Higher DNA concentrations decrease amplicon specificity (i.e., extra bands are more likely), particularly when a large number of cycles are employed.
• Use the higher DNA concentrations when fewer cycles are desired (e.g. to increase fidelity).

Primers

• Generally 20-30 nucleotides in length.
• Ideal GC content is 40-60%.
• Space GC residues evenly within the primer.
• Primer pairs should have Tms within 5°C of each other.
• Avoid secondary structure (i.e., hairpins) within each primer and potential dimerization between the primers present.
• When engineering sites into the end of primers, 4-6 extra bases should be added 5´ to the site.
• Final concentration should be 0.05-1 µM, typically 0.1-0.5 µM of each primer.
• Higher concentrations may increase secondary priming and create spurious amplification products.

Magnesium Concentration

• 1.5-2.0 mM is optimal for Taq DNA Polymerase, but the ideal concentration depends on template, buffer, DNA and dNTPs (each has the potential to chelate magnesium).
• If [Mg2+] is too low, no PCR product will be seen.
• If [Mg2+] is too high, undesired PCR products may be seen.

Deoxyribonucleotide triphosphates (dNTPs) >

• Typical concentration is 200 µM of each dNTP.
• 50-100 µM enhances fidelity of polymerization, but reduces yields.
• Higher concentrations increase yields particularly in long PCR, but can reduce fidelity.

DNA Polymerase

• The choice of the correct polymerase depends among other things on the purpose as well as on the template used (standard PCR: Taq DNA Polymerase S > with high accuracy, Taq Polymerase E > with high yield; master mixes: Standard PCR master mix > or RedMasterMix > with red dye).
• For multiplex PCR, there exist special multiplex master mixes >.
• Hot start applications are recommended to increase specificity or if you use difficult templates. For this purpose there exist special Hot Start Polymerases M3307 > or M3006 >.
• For PCRs for the purpose of cloning or other procedures requiring a low error rate, there are thermostable high fidelity proofing polymerases such as Pfu >, AQ97>, or ReproFast >. These enzymes make far fewer errors during amplification and increase the chances of an amplicon without mistakes.
•  For genotyping and other applications where a high discrimination rate is required, there is a new highly selective DNA polymerase, SNP Pol DNA Polymerase >. It specifically distinguishes mismatched primer-template complexes and ony produces specific amplicons in case of perfectly matched primer pairs.
• For the real-time quantitative PCR there exist highly specialized qPCR master mixes >. Hereby, the choice of the correct master mix depends on the qPCR device you are using. Alternatively, you can simply use a universal master mix, such as the GNX Versa qPCR MasterMix 2X > from Genaxxon, which automatically ‘adjusts’ the correct amount of ROX for the respective instrument. Even if there are different PCR cyclers in the laboratory, you no longer need to worry about which master mix to use
• The amount of DNA polymerase used in the PCR reaction can significantly influence the PCR result (use 1.25-1.5 units Taq Polymerase > for a 50μL volume).

General Guidelines

Annealing Temperature and Duration

• Match the Tms within 5°C of each other.
• Typical annealing temperatures are 5°C below the lowest primer's Tm and often fall in the range of 50-60°C.
• Test higher annealing temperatures if spurious amplification products are observed.
• Typical annealing times are 15-30 seconds.

Extension Time

• Extensions are normally performed at 68°C.
• In general, use extension times of one minute per 1000 base (1 kb) pairs (e.g. 3 minutes for a 3 kb product). 
• For products less than 1 kb, use 45-60 seconds.
• Products greater than 3 kb, or reactions using more than 30 cycles, may require longer extensions.

The amplification of templates with high GC content, strong secondary structures, low concentrations or amplicons more than 5 kb often require optimization of the PCR conditions. Typically, 15-30 seconds of denaturation should be performed at 95 ° C during PCR.