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 |
Melting curve analysis is performed on target samples in order to determine PCR reaction efficiency, product Tm, primer-dimer product Tm and acquisition temperature. Reaction optimization is carried out for target primers and MgCl2 concentration.Quantitative real-time RT-PCR is then performed with the addition of a signal acquisition temperature step 2°C lower than target product Tm. This step increases the specificity of the reaction eliminating nonspecific product accumulation signal.
Amplification parameters include an initial denaturation step followed by 40-45 amplification cycles. Cycles consist of denaturation, annealing, extension and signal acquisition steps. A cooling step follows amplification.