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Efficient and Reliable PCR Setup Using Eppendorf MasterMix

bjective was to test the reproducibility of the results and to ascertain the stability of the MasterMix at different storage and application temperatures. Tests were carried out on different types of applications and different individual applications.

Materials and Methods

Experiment 1:
Reproducibility of parallel PCR preparations tested using Eppendorf MasterMix
In 56 parallel preparations, a 400-bp fragment from the gene of the Taq polymerase (60% GC content) was amplified. 15 l template/primer mix was added to 10 l of 2.5 x MasterMix (per preparation: 1.5 mM MgCl2, 200 M each of dNTP, 1.25 U Taq; 200 pg pTTQ18 plasmid DNA, 20 pmol per primer). The reaction took place in a 0.2 ml PCR tube with the following PCR program on Mastercycler gradient** from Eppendorf.

94C 3 minute initial denaturation
30 cycles:
94C 15 seconds denaturation
59C 10 seconds annealing
72C 40 seconds extension

The fragments were separated by electrophoresis in a 1.2% agarose gel in TAE buffer at 110 V.

Experiment 2:
Stability test for PCR MasterMix: Using Eppendorf MasterMix after storage under different condition temperatures in three different PCR systems.
Following incubation for 48 hours each at 20C, 28C, 2028C or 37C, Eppendorf MasterMix was tested in three different PCR systems. According to the system used, a 360-bp fragment from the human gene apolipoprotein B (Exon 26), a 547-bp fragment with the coding sequence of the pyrophosphatase gene of Thermo thermophilus (70% GC content) and a 10-kb fragment from the genome of the phage lambda was amplified. The target DNA
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