Thermal Cycler Temperature Variation and Its Effect on the Polymerase Chain Reaction
Abstract
This study was undertaken to investigate the source of variation in Polymerase Chain Reaction (PCR) amplification assays that we have encountered periodically in our studies. Two approaches, namely, PCR/agarose gel analysis and thermal probe analysis, were used in this investigation. PCR/agarose gel analysis demonstrated random variation in the quantity and quality of amplified product from well to well, both within and between trials. Several modifications to the procedure did not eliminate variability. Thermal probe analysis indicated significant well to well temperature viability among certain wells or groups of wells within trial (p = 0.01) and significant temperature viability among certain well positions from one trial to another (p = 0.05). Thermal probe analysis also indicated large differences between the programmed setpoint temperatures and the actual temperatures inside the tubes for all three PCR events at the beginning of the soak period and two of three PCR events at the end of the soak period. The data from this investigation and other studies leads to the conclusion that the source of variability in PCR amplification efficiency we have experienced is most likely due to the inherent inability of our thermal cycler to maintain consistent temperature homogeneity across the heating block during PCR amplification reactions. It is advisable that replicate samples from amplification be prepared since a particular well cannot be expected to provide consistent results from well to well within a trial or from one trial to the next.
Document Details
- Document Type
- Technical Report
- Publication Date
- Mar 01, 1993
- Accession Number
- ADA264660
Entities
People
- Doug E. Bader
Organizations
- Defence Research and Development Canada