Transferman nk2

Microdissection was performed in an Eppendorf TransferMan NK2 micromanipulator coupled to a Zeiss Axiovert 100 microscope. We microdissected ten copies of a complete metacentric B chromosome (Bm) for DNA sequencing (µB-DNA). Moreover, two different B-derived DNA probes were generated through microdissection for FISH experiments: 1) one arm of one copies of the Bm chromosome (µBm-probe) (in order to test its isochromosome nature) and 2) ten copies of a complete submetacentric B chromosome (Bsm) (µBsm-probe). The microdissected DNAs were placed in 9 µl of DNase-free ultrapure water and then amplified using the GenomePlex Single Cell Whole Genome Amplification Kit (wga4-Sigma) [53] (link). After the initial amplification, we generated DNA probes (µBm-probe and µBsm-probe) labelled with digoxigenin-11-dUTP (Roche Applied Science) using the GenomePlex Whole Genome Amplification Reamplification Kit (wga3-Sigma) following the manufacturer's protocol.

Chromosome microdissection was performed in an Eppendorf TransferMan NK2 micromanipulator coupled with a Zeiss Axiovert 100 microscope. For chromosome PageBreakpainting, ten B chromosomes were microdissected from the cytogenetic preparations of the samples from each species (Characidiumalipioi, Characidiumgomesi and Characidiumoiticicai) carrying one extra chromosome. The probes for Characidiumalipioi, Characidiumgomesi and Characidiumoiticicai denoted CaB, CgB and CoB, respectively.
Microdissected DNA from each species was placed into a tube containing 9 µL of DNase-free ultrapure water and amplified using the GenomePlex Single Cell Whole Genome Amplification Kit (wga4 Sigma) (Gribble et al. 2004 (link)). After the initial amplification, the DNA probes CaB, CgB and CoB were generated and were then labeled with Digoxigenin-11-dUTP (Roche Applied Science) using the GenomePlex Whole Genome Amplification Reamplification Kit (wga3 Sigma) according to the manufacturer’s protocol.