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HotStarTaq PCR Handbook

DNA and viral nucleic acids, and the RNeasy system for RNA preparation from a variety of sources. For more information about QIAprep, QIAamp, DNeasy, and RNeasy products, contact one of our Technical Service Departments (see inside front cover).

Quantity of starting template

The annealing efficiency of primer to template is an important factor in PCR. Owing to the thermodynamic nature of the reaction, the primer:template ratio strongly influences the specificity and efficiency of PCR and should be optimized empirically. If too little template is used, primers may not be able to find their complementary sequences. Too much template may lead to an increase in mispriming events. As an initial guide, spectrophotometric and molar conversion values for different nucleic acid templates are listed in Tables 5 and 6 respectively.

Table 5. Spectrophotometric conversions for nucleic acid templates


Absorbance at 260 nm = 1

Table 6. Molar conversions for nucleic acid templates


* Base pairs in haploid genome
For single-copy genes

2. Primer design, concentration, and storage
Standard PCR primers

Prerequisites for successful PCR include the design of optimal primer pairs, the use of appropriate primer concentrations, and the correct storage of primer solutions. Some general guidelines are given in Table 7


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