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Efficient and Reliable PCR Setup Using Eppendorf MasterMix

10 ng T. thermophilus HB8 genomic DNA or 25 ng lambda DNA; 20 pmol per primer). The reaction took place in a 0.2 ml PCR tube with the following PCR program on the Mastercycler gradient:

Human genomic DNA
94C 3 minute initial denaturation
35 cycles:
94C 20 seconds denaturation
52C 10 seconds annealing
72C 25 seconds extension

T.th genomic DNA
95C 3 minute initial denaturation
35 cycles:
94C 20 seconds denaturation
55C 10 seconds annealing
72C 40 seconds extension

Lambda DNA
93C 3 minute initial denaturation
20 cycles:
93C 15 seconds denaturation
62C 30 seconds annealing
68C 12 seconds extension

Plasmid DNA (pTTQ Taq)
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 70 V.

Results

Experiment 1:
As shown in Fig. 1, the same product was amplified in all PCR preparations, with the bands all showing the same degree of intensity. MasterMix is an easy-to-handle, timesaving method of guaranteeing optimal reproducibility from one experiment to the next.


Fig. 1: Reproducibility of parallel PCR preparations with MasterMix A 400-bp fragment from the gene of Taq DNA polymerase was amplified using MasterMix.

Experiment 2:
As shown in Fig. 2, pre-incubating MasterMix for 4
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Source:


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