( was used to amplify the 18 kb target. ...)TripleMaster PCR SystemThe Ideal Choice for Long-Range PCR

was used to amplify the 18 kb target. Primers specific to the human Factor VIII gene and the human tPA gene were synthesized by Integrated DNA Technologies or Sigma-Genosys (see Table 1). All PCR reagents were used at the final concentrations recommended by the manufacturer (see Table 2). Genomic DNA was isolated from whole blood preserved in EDTA using the Eppendorf Perfect gDNA Blood Mini Kit. For all instances in which a competitors PCR system did not contain a necessary reagent, such as molecular biology grade water or dNTP's, the Eppendorf version of that reagent was used. All thermal cycling was performed on an Eppendorf Mastercycler Gradient using optimized parameters for the specific primer/template system (see Table 3). Samples were analyzed by field inversion gel electrophoresis in a 0.5x TBE buffer using a 180 V forward voltage, a 120 V reverse voltage, and a switch time ramp of 0.1 0.8 seconds (linear shape). 15 ml of the 50 ml reactions were loaded on a 1.0% agarose gel, run for 16 hours, stained in a 0.5 mg/ml ethidium bromide solution and photographed.

Table 1: Primers sequences for long-range PCR targets.

Primer Sequence Factor VIII Forward 5' ACC CAC CAG TCT TGA AAC GC 3' Factor VIII Reverse 5' GGT GCT CCA GGC ATT GAT TG 3' tPA Forward 5' CCT TCA CTG TCT GCC TAA CTC CTT CGT GTG TTC C 3' tPA 24 kb Reverse 5' TGT CTC CAG CAC ACA GCA TGT TGT CGG TGA C 3' tPA 18 kb Reverse 5' GCA GGG TCT GCA GAA CTC TGA GCT GTA CT
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