Lars E. Peters, Walter Gross, and Jennifer Diers
Eppendorf 5 Prime, Inc., Boulder, CO, USA This experiment was performed to compare the Eppendorf TripleMaster PCR System with the long-range PCR products of leading competitors. Eppendorf was compared to competitors T and R in genomic DNA amplification; and competitors T, R, and AB in Lambda DNA amplification. Very stringent cycling conditions were used for all products in terms of a short extension time and low number of cycles. Please note: The cycling conditions are not according to the manufacturers instructions. These conditions prove the high processivity of the TripleMaster polymerase when tested against competitor products. In order to gain the success seen with the TripleMaster system, usual cycling conditions for competitors products would require much longer extension times and many more cycles.

Experimental Setup

Genomic DNA amplification

Reactions were executed using 200 ng of commercially purchased human gDNA (Roche). Primers targeting a 24 kb region of the human tPA gene were used. Primer sequences for the 24 kb human tPA universal forward and the 24 kb reverse primers may be found in the Eppendorf TripleMaster PCR System manual. Reaction components from each manufacturer were used as follows in the concentrations presented in parentheses:

Eppendorf TripleMaster PCR System

10X Tuning Buffer (1X)
Eppendorf dNTP Mix (0.5 mM)
MgCl2 (2.4 mM)
TripleMaster (2.0 U)
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