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Optimizing pfuturbo DNA Polymerase Amplification Reactions with,,,Perfect Match PCR Enhancer

amplified from human genomic DNA and the 6.2- and 6.6-kb fragments were amplified from Big Blue transgenic mouse genomic DNA that contained 40 copies of lambda phage DNA per genome.

PCR reactions were conducted in a Stratagene RoboCycler Gradient 96 temperature cycler fitted with a Hot Top Assembly, using 200-l thin-walled PCR tubes. Temperature cycling parameters were devised for the 120-bp target: 1 cycle at 94 for 1 minute, followed by 30 cycles at 94C for 1 minute (denaturation); 54C for 1 minute (annealing) and 72C for 1 minute (extension); and 1 final extension cycle of 72C for 10 minutes. The amplified reaction products were electrophoresed on a 4 to 20% TBE/acrylamide gel, stained with ethidium bromide, and imaged using the Eagle Eye II still video system. Temperature cycling parameters were established for the 6.2-kb target: 1 cycle at 95C for 1 minute, followed by 30 cycles at 95C for 1 minute (denaturation); 58C for 1 minute (annealing) and 72C for 7 minutes (extension); and 1 final extension cycle of 72C for 10 minutes. The same cycling conditions were used for the 6.6-kb target, except we employed a 60C annealing temperature. For the long targets, the amplified reaction products were electrophoresed on a 1% agarose/1X TBE gel.

Difficult PCR Amplification Systems

Figure 1

To demonstrate the variety of improvements Perfect Match PCR enhancer can have on PCR performance, we selected primer-template systems which generate multiple bands (Figure 1 and Figure 3) or bands of the wrong size (Figure 2).

Figure 2

Improved Product Yield and Specificity

The first system tested was designed to amplify a 6.2-kb fragment from mouse genomic DNA. In the absence of Perfe
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