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Real-Time PCR: General Considerations, Rev A

mized primer set for a quantitative real-time assay.


Considerations for General PCR Optimization
General Laboratory Practices for Quantitative Real-Time PCR
In general, follow these practices to ensure the highest probability of success:
Wear gloves
Use screwcap tubes
Use aerosol-resistant filter tips
Use calibrated pipets dedicated to PCR
Use PCR-grade water and use only for PCR
Use a no-template control to verify absence of contamination
Prepare reactions in replicate ideally as triplicates

Replicate Quality
To obtain good replicates, a master mix should be prepared with all reaction components including the sample Use a hot-start enzyme to prevent nonspecific amplification during preparation
Make up a master mix with sufficient volume to prepare all replicate samples
Pipet once per well


DNA Source
The source of the template affects the accessibility of the target sequence and must be considered during optimization. It is important to optimize the reaction for the template concentrations that will be used in your experiment.

Genomic DNA (Intact, High Molecular Weight DNA)
Cut with a restriction enzyme that does not cut within the region to be amplified
Boil DNA stock for 10 min and place immediately on ice

Plasmid DNA
If there are problems with amplification, linearize the plasmid with a restriction enzyme that does not cut within the target

cDNA
RNA must be free from genomic DNA contamination treat with RNase-free DNase prior to reverse tr
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