( centration. The GC-RICH Resolution Solu...)Using the TripleMaster PCR System for Robust Amplification of ,,, GC-Rich DNA Templates

centration. The GC-RICH Resolution Solution of the Roche system was tested at concentrations of 0 2 M in 0.5 M increments, as stated in the user manual. All amplifications were performed on an Eppendorf Mastercycler Gradient (see Table 3). Samples were analyzed by standard agarose gel electrophoresis in a 1x TBE buffer. 5 ml of the 50 ml reactions were loaded on a 2.0% agarose gel, run at 6 V/cm, stained in a 0.5 mg/ml ethidium bromide solution and photographed with a CCD camera.

In experiment 1, the TripleMaster PCR System was used to determine the optimal reaction mix and thermal cycling conditions for the apolipoprotein E and retinoblastoma targets. Reactions were then set up in parallel to compare the TripleMaster PCR System with the leading suppliers.

In experiment 2, the TripleMaster PCR System was used to determine the reaction mix conditions that generated the highest product yield for the MC1R target using standard thermal cycling parameters (95C for 5 minutes, followed by 35 cycles at 95C for 30 seconds, 56C for 30 seconds, and 72C for 1 minute). Touchdown PCR was then employed to prove this method is capable of achieving optimal amplification and specificity without the need for thermal cycling optimization experiments. Reactions were set up in parallel to compare the performance of the TripleMaster PCR System with the performance of the leading suppliers.

Table 1: Primer sequences, amplicon size and GC content for GC-rich PCR targets.

Target Amplicon % GC Primer Sequences Human retinoblastoma gene
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