This facility was established by a grant from
Health Resources and Services Administration (HRSA)
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Rapid Cycle Real-Time (kinetic) PCR using LightCycler instumentation (Roche)
Introduction / Quantitative RT-PCR / PCR Primer Design / Sample preparation / Amplification Data Analysis / References / Services and Fees / RT-PCR Services Request Form |
Suitable PCR primer pairs are designed with the LightCycler Probe Design Software
The main criteria for primer design are length, sequence and melting temperature. Recommended primer length is 18-24 nucleotides. GC content of the primers should range between 40% and 60%. The most important region for the specific priming is the 3’ region of the primer; amplification starts here. The 3’ ends should be free of secondary structures, repetitive sequences, palindromes and highly degenerate sequences. The sequences should lack complementarity to each other, especially at their 3’ ends (so primer-dimer will not form). Most primers should have melting emperatures between 55∞C and 65∞C. Primers that are used together should have similar Tm values.