Figure 3

Multiplex PCR analysis of clones obtained after co-transformation of nine overlapping fragments in S . cerevisiae and of clones obtained from control experiments. The multiplex primer mix was designed to produce nine amplicons, ranging in size from 119–516 bp. Each amplicon covered a specific SHR-sequence. Amplicons were separated on a 2% agarose gel by electrophoresis. Lanes 1–5 represent clones obtained after transformation of a full set of fragments. As a negative control genomic DNA of CEN.PK113-5D was used (−); The later fully analyzed plasmid pUDC074 is added as a positive control (+). All nine bands were obtained in clones 1–5. The clones obtained from transformation of an incomplete mix show a completely different multiplex pattern (#1 and #2). In the lanes labeled ‘L’ a 50 bp GeneRuler ladder was loaded; sizes are indicated. In total 40 clones were analyzed and 38 multiplex patterns matched the positive control.