THERMAL CYCLER TEMPERATURE VARIATION AND ITS EFFECT ON THE POLYMERASE CHAIN REACTION

Authors
  1. Bader, D.E.
Corporate Authors
Defence Research Establishment Suffield, Ralston ALTA (CAN)
Abstract
The 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 emplified 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 variability among certain wells or groups of wells within a trial (p=0.01) and significant temperature variability among certain well positions from one trial to another (p=0.01). 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. TRUNCATED
Keywords
Polymerase Chain Reaction;Cloning (genetics)
Report Number
DRES-583 —
Date of publication
01 Mar 1993
Number of Pages
49
DSTKIM No
93-01652
CANDIS No
129865
Format(s):
Hardcopy;Originator's fiche received by DSIS

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