Primer ConcentrationThe recommended primer concentration for PCR is between 0.1μM and 1μM of each primer. The use of higher concentrations of primers can have the following effects:
- If the primers are capable of forming dimers, raising their concentration only results in the creation of primer-dimers and does not improve the amplification of the desired PCR product. Primer-derived oligomers will possibly contaminate the reaction.
- If the primers do not form primer-dimers, it is likely that raising the primer concentration will lead to non-specific primer binding and the creation of spurious, undesirable PCR products.
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However, to amplify short PCR target sequences, careful calculation of the optimum primer concentration is required. For example, if the target fragment length is 100bp, a greater number of PCR product molecules is required to provide a specified amount of amplified DNA (in nanograms) than for a larger target fragment. In order to generate the required number of PCR product molecules, a greater number of primers may be needed. Therefore, concentration of primers higher than 1μM may be necessary, and desirable, for short target sequences.
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