For PCR comparisons, amplifications (50 l) employed 200 M (<1 kb) or 500 M (>17 kb) each dNTP and 100 ng (<1 kb) or 200 ng (>17 kb) of each primer. The following amounts of DNA template were used: 100 ng (<1-kb targets) or 240 ng (17-kb and 30-kb targets) of genomic DNA or 15 ng (48 kb) of lambda DNA. As recommended by each enzyme manufacturer, amplifications were carried out with 2.5 U (<1 kb) or 5 U (>17 kb) of the Herculase formulation, 2.5 U of Platinum Taq DNA polymerase High Fidelity, or 2.6 U of the Expand 20 kbPlus blend. The following amounts of DMSO were added to Herculase PCRs: 0% (0.1-kb to 17-kb targets), 3% (30-kb target), or 7% (48-kb target).
Reactions were cycled in 200-l thin-walled PCR tubes using a RoboCycler gradient 96 temperature cycler (Stratagene)(<1-kb targets) or a single-block thermocycler (>17-kb targets) fitted with a Hot Top Assembly. For enzyme comparisons, identical temperature cycling parameters were employed that consisted of (<1-kb targets) 1 cycle at 94C for 1 minutes, followed by 30 cycles at 94C for 40 seconds (denaturation), 58C for 40 seconds (annealing) and 68C for 1 minute, or (>17-kb targets) 1 cycle at 92C for 2 minutes, followed by 10 cycles at 92C for 10 seconds (denaturation), 60 to 65C for 30 seconds (annealing) and 68C for 1 minute per kb of target, followed by 20 cycles at 92C for 10 seconds (denaturation), 60 to 65C for 30 seconds (annealing) and 68C for 1 minute per kb of target plus an additional 10 seconds/cycle. After 30 cycles, one final extension cycle of 68C for 10 minutes was carried out.
PCR products le