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Efficient and Reliable PCR Setup Using Eppendorf MasterMix

used was human genomic DNA, T.th genomic DNA or lambda DNA. The PCR reaction volumes were each 50 l. 30 l template/primer mix was added to 20 l of 2.5 x MasterMix (per preparation: 1.5 mM MgCl2, 200 M each of dNTP, 1.25 U Taq; 20 ng human genomic DNA, 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 programs 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

The fragments were separated by electrophoresis in a 1.2% agarose gel in TAE buffer at 70 V.

Experiment 3:
Application spectrum of Eppendorf MasterMix regarding target fragment length and template

The area of use of MasterMix was tested by amplifying fragments of different lengths (160 bp10 kb). Four different templates were used: human genomic DNA, T. thermophilus HB8 genomic DNA, plasmid DNA (pTTQ Taq) and lambda DNA.

The PCR reaction volumes were each 50 l. 30 l template/primer mix was added to 20 l of 2.5 x MasterMix (per preparation: 1.5 mM MgCl2, 200 M each of dNTP, 1.25 U Taq; 20 ng human genomic DNA,
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