( Eppendorf TripleMaster PCR S...)Using the TripleMaster PCR System for Robust Amplification of ,,, GC-Rich DNA Templates

Eppendorf TripleMaster PCR System
Invitrogen ThermalAce DNA Polymerase Kit
Roche GC-RICH PCR System
Genomic DNA isolated using the Eppendorf Perfect gDNA Blood Mini Kit
Eppendorf Mastercycler Gradient Thermal Cycler
EasyCast Horizontal Minigel System (Owl Separation Systems)
SeaKem LE Agarose (BioWhittaker Molecular Applications)
1x TBE (Eppendorf)
Ethidium Bromide (Sigma)
Alpha Imager CCD Camera Documentation System (Alpha Innotech Corporation)

Methods

The performance of the TripleMaster PCR System for GC-rich PCR was tested against other PCR systems specifically designed for GC-rich PCR. Three different DNA sequences with high GC-content were chosen for amplification. The primer sequences, length of the expected PCR products and percentage of GC content in the amplified sequences are listed in Table 1. Genomic DNA was isolated from whole blood preserved in EDTA using the Eppendorf Perfect gDNA Blood Mini Kit. The oligonucleotide primers were obtained from Sigma-Genosys. Triplicate reactions were prepared in accordance with the manufacturers recommended specifications and all PCR reagents were used at final concentrations within the ranges recommended by the manufacturer (see Table 2). The 1.6x Eppendorf Tuning Buffer concentration (with 4.0 mM Mg2+) was used for the apolipoprotein E and retinoblastoma gene reactions due to the size of the targets. The TripleMaster PCR System user manual recommends a higher buffer concentration for targets less than 500 bp because the Tuning Buffer is a low-salt buffer specifically designed for long-range PCR, and short targets require a buffer with a higher salt con
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